Mutant lysenin pores

ABSTRACT

The invention relates to mutant forms of lysenin. The invention also relates to analyte characterisation using lysenin.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/391,660 filed Oct. 9, 2014, which is a national stage filing under 35U.S.C. 371 of PCT International Application No. PCT/GB2013/050667 filedMar. 15, 2013, which claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/622,174 filed Apr. 10, 2012. Thecontents of each of the above referenced applications are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to mutant forms of lysenin. The invention alsorelates to analyte characterisation using the mutant forms of lysenin.

BACKGROUND OF THE INVENTION

Nanopore sensing is an approach to sensing that relies on theobservation of individual binding or interaction events between analytemolecules and a receptor. Nanopore sensors can be created by placing asingle pore of nanometer dimensions in an insulating membrane andmeasuring voltage-driven ionic transport through the pore in thepresence of analyte molecules. The identity of an analyte is revealedthrough its distinctive current signature, notably the duration andextent of current block and the variance of current levels.

There is currently a need for rapid and cheap nucleic acid (e.g. DNA orRNA) sequencing technologies across a wide range of applications.Existing technologies are slow and expensive mainly because they rely onamplification techniques to produce large volumes of nucleic acid andrequire a high quantity of specialist fluorescent chemicals for signaldetection. Nanopore sensing has the potential to provide rapid and cheapnucleic acid sequencing by reducing the quantity of nucleotide andreagents required.

Two of the essential components of sequencing nucleic acids usingnanopore sensing are (1) the control of nucleic acid movement throughthe pore and (2) the discrimination of nucleotides as the nucleic acidpolymer is moved through the pore. In the past, to achieve nucleotidediscrimination the nucleic acid has been passed through a mutant ofhemolysin. This has provided current signatures that have been shown tobe sequence dependent. It has also been shown that a large number ofnucleotides contribute to the observed current when a hemolysin pore isused, making a direct relationship between observed current andpolynucleotide challenging.

While the current range for nucleotide discrimination has been improvedthrough mutation of the hemolysin pore, a sequencing system would havehigher performance if the current differences between nucleotides couldbe improved further. In addition, it has been observed that when thenucleic acids are moved through a pore, some current states show highvariance. It has also been shown that some mutant hemolysin poresexhibit higher variance than others. While the variance of these statesmay contain sequence specific information, it is desirable to producepores that have low variance to simplify the system. It is alsodesirable to reduce the number of nucleotides that contribute to theobserved current.

Lysenin (also known as efL1) is a pore-forming toxin purified from thecoelomic fluid of the earthworm Eisenia fetida. It specifically binds tosphingomyelin, which inhibits lysenin-induced hemolysis (Yarnaii et al.,J. Biol. Chem. 1998; 273(9): 5300-6). The crystal structure of lyseninis disclosed in De Colbis et al. Structure, 2012; 20: 1498-1507.

SUMMARY OF THE INVENTION

The inventors have surprisingly identified a region within the lyseninmonomer which can be modified to alter the interaction between themonomer and a polynucleotide. This region corresponds to from aboutposition 44 to about position 126 of SEQ ID NO: 2. The inventionconcerns mutant monomers in which one or more modifications have beenmade to the identified region to improve the ability of the monomer tointeract with a polynucleotide. The inventors have also surprisinglydemonstrated that pores comprising the novel mutant monomers have anenhanced ability to interact with polynucleotides and therefore displayimproved properties for estimating the characteristics of, such as thesequence of, polynucleotides. The mutant pores surprisingly displayimproved nucleotide discrimination. In particular, the mutant poressurprisingly display an increased current range, which makes it easierto discriminate between different nucleotides, and a reduced variance ofstates, which increases the signal-to-noise ratio. In addition, thenumber of nucleotides contributing to the current as the polynucleotidemoves through the pore is decreased. This makes it easier to identify adirect relationship between the observed current as the polynucleotidemoves through the pore and the polynucleotide.

Accordingly, the invention provides a mutant lysenin monomer comprisinga variant of the sequence shown in SEQ ID NO: 2, wherein the monomer iscapable of forming a pore and wherein the variant comprises one or moremodifications within the region of from about position 44 to aboutposition 126 of SEQ ID NO: 2 which alter the ability of the monomer tointeract with a polynucleotide.

The invention also provides:

-   -   a construct comprising two or more covalently attached monomers        derived from lysenin, wherein at least one of the monomers is a        mutant lysenin monomer of the invention;    -   a polynucleotide which encodes a mutant lysenin monomer of the        invention or a genetically fused construct of the invention;    -   a homo-oligomeric pore derived from lysenin comprising a        sufficient number of mutant lysenin monomers of the invention;    -   a hetero-oligometic pore derived from lysenin comprising at        least one mutant lysenin monomer of the invention;    -   a pore comprising at least one construct of the invention;    -   a method of characterising a target analyte, comprising: (a)        contacting the target polynucleotide with a pore of the        invention such that the target polynucleotide moves through the        pore; and (b) taking one or more measurements as the analyte        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the target analyte        and thereby characterising the target analyte;    -   a method of forming a sensor for characterising a target        polynucleotide, comprising forming a complex between a pore of        the invention and a polynucleotide binding protein and thereby        forming a sensor for characterising the target polynucleotide;    -   a sensor for characterising a target polynucleotide, comprising        a complex between a pore of the invention and a polynucleotide        binding protein;    -   use of a pore of the invention to characterise a target analyte;    -   a kit for characterising a target polynucleotide comprising (a)        a pore of the invention and (b) a polynucleotide binding        protein;    -   an apparatus for characterising target polynucleotides in a        sample, comprising (a) a plurality of pores of the invention        and (b) a plurality of polynucleotide binding proteins;    -   a method of improving the ability of a lysenin monomer        comprising the sequence shown in SEQ ID NO: 2 to characterise a        polynucleotide, comprising making one or more modifications        within the region of from about position 44 to about position        126 of SEQ ID NO: 2 which alter the ability of the monomer to        interact with a polynucleotide and do not affect the ability of        the monomer to form a pore;    -   a method of producing a construct of the invention, comprising        covalently attaching at least one mutant lysenin monomer of the        invention to one or more monomers derived from lysenin; and    -   a method of forming a pore of the invention, comprising allowing        at least one mutant monomer of the invention or at least one        construct of the invention to oligomerise with a sufficient        number of monomers of the invention, constructs of the invention        or monomers derived from lysenin to form a pore.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an example schematic of the use of a helicase (labelled A)to control DNA movement through a lysenin nanopore (labelled B). 1) AssDNA substrate with an annealed primer containing a cholesterol-tag(labelled D) is added to the cis side (labelled X) of the bilayer(labelled C). The cholesterol tag binds to the bilayer, enriching thesubstrate at the bilayer surface. 2) Helicase added to the ciscompartment binds to the DNA. In the presence of divalent metal ions andNTP substrate, the helicase moves along the DNA (grey arrow). 3) Underan applied voltage, the DNA substrate is captured by the nanopore viathe leader section on the DNA. The DNA is pulled through the pore underthe force of the applied potential until a helicase, bound to the DNA,contacts the top of the pore, preventing further uncontrolled DNAtranslocation. During this process dsDNA sections (such as the primer)are removed. The helicase movement along the DNA in a 3′ to 5′ directionpulls the threaded DNA (direction of DNA movement shown with a blackarrow) out of the pore against the applied field. 4) The helicase pullsthe DNA out of the nanopore, feeding it back to the cis compartment. Thelast section of DNA to pass through the nanopore is the 5′-leader. 5)When the helicase moves the DNA out of the nanopore it is lost back tothe cis compartment. Alternatively, if the DNA is captured by the 3′end, then the DNA will move through the pore from cis to trans (labelledY) under control of the helicase, finally exiting on the trans side ofthe bilayer.

FIG. 2 shows the DNA substrate design used in Examples 1, 2, 3, 4, 5 and6. The DNA substrate consists of a 400 base section of ssDNA from PhiX(SEQ ID NO: 13, labelled A), with a 50T 5′-leader (indicated by thedashed region of strand A). Annealed to this strand just after the 50Tleader is a primer (labelled B) containing a 3′ cholesterol tag(labelled C) to enrich the DNA on the surface of the bilayer, and thusimprove capture efficiency.

FIG. 3 shows a current trace (y-axis=current (pA), x-axis=time (min) forA and B) of a wild-type lysenin pore inserted into a DPhPC bilayer. A)shows a stable open pore current of approximately +280 pA was observedat +120 mV (625 mM KCl, 100 mM Hepes, pH 8.0, 75 mM PotassiumFerrocyanide (II), 25 mM Potassium Ferricyanide (III), 10 mM MgCl₂,wild-type lysenin (SEQ ID NO: 2)) in the absence of DNA and helicase. B)Shows that upon the addition of DNA, helicase and ATP (0.3 nM 400 merDNA (SEQ ID NO: 13 and 14), Hel308 Mbu, (100 nM, SEQ ID NO: 15), 1 mMATP) there is no clear DNA capture, and no helicase controlled DNAmovement through the nanopore.

FIG. 4 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore (lysenin-E84D/E85K, SEQ ID NO: 2 withmutations E84D/E85K) in a controlled fashion, producing stepwise changesin current as the DNA moves through the nanopore. A) Shows an examplecurrent trace (y-axis=current (pA), x-axis=time (min)) of DNA captureand Hel308 Mbu controlled 400 mer DNA movement, observed as lowercurrent blocks at ˜200 pA from the open-pore level of ˜400 pA (180 mV,625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II), 25 mMpotassium ferricyanide, 0.3 nM 400 mer DNA (SEQ ID NO: 13 and 14), 100nM Hel308 Mbu (SEQ ID NO: 15), 1 mM ATP, 10 mM MgCl₂, Lysenin-E84D/E85K(SEQ ID NO: 2 with mutations E84D/E85K)). The star indicates helicasecontrolled DNA movement, Under an applied potential DNA with helicasebound is captured by the lysenin nanopore. This produces blocks incurrent from the open-pore level (˜400 pA) to a DNA level (˜220 pA). B)Shows an expanded view (y-axis=current (pA), x-axis=time (min)) of thehelicase controlled DNA movement in the upper trace. The DNA level showsstepwise changes in current as the enzyme moves the DNA through thepore.

FIG. 5 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore (Lysenin-E92N/E94N/E97N/D121N/D126N, SEQ IDNO: 2 with mutations E92N/E97N/E97N/D121N/D126N) in a controlledfashion, producing stepwise changes in current as the DNA moves throughthe nanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time(min)) of typical Hel308 Mbu controlled 400 mer DNA movements(120 mV, 625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassiumferrocyanide(II), 25 mM potassium ferricyanide, 0.3 nM 400 mer DNA (SEQNO: 13 and 14), 100 nM Hel308 Mbu (SEQ ID NO: 15), 1 mM ATP, 10 mMMgCl₂, Lysenin-E92N/E94N/E97N/D121N/D126N (SEQ ID NO: 2 with mutationsE92N/E94N/E97N/D121N/D126N)). Under an applied potential DNA is capturedby the lysenin nanopore. This lysenin mutant shows a high level of DNAcapture vs. the WT lysenin. DNA captured in the pore produces blocks incurrent from the open-pore level (˜280 pA) to a DNA level (˜110 pA). DNAwith helicase bound shows stepwise changes in current as the enzymemoves the DNA through the pore. Helicase controlled DNA movements aremarked by a star. B) An expanded view of one of the typical helicasecontrolled DNA movements (y-axis=current (pA), x-axis=time (min) in theupper trace. The DNA level shows stepwise changes in current as theenzyme moves the DNA through the pore.

FIG. 6 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore (Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A, SEQID NO: 2 with mutations E84Q/E85K/E92Q/E97S/D126G/E167A) in a controlledfashion, producing stepwise changes in current as the DNA moves throughthe nanopore A) Shows an example current trace (y-axis=current (pA),x-axis=time (min)) of typical Hel308 Mbu controlled DNA movements (180mV, 625 mM KCl, 100 Hepes pH 8.0, 75 mM potassium ferrocyanide(II), 25mM potassium ferricyanide, 0.6 nM 400 mer DNA (SEQ ID NO: 13 and 14),100 nM Hel1308 Mbu (SEQ ID NO: 15), 1 mM ATP, 10 mM MgCl₂,Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A, (SEQ ID NO: 2 with mutationsE84Q/E85K/E92Q/E97S/D126G/E167A)). Under an applied potential DNA iscaptured by the lysenin nanopore. This lysenin mutant shows a high levelof DNA captures, the WT lysenin. The DNA captured in the pore producesblocks in current from the open-pore level (˜390 pA) to a DNA level(˜200 pA). DNA with helicase bound shows stepwise changes in current asthe enzyme moves the DNA through the pore. Helicase controlled DNAmovements are marked by a star. B) An expanded view of one of thetypical helicase controlled DNA movements (y-axis=current (pA),x-axis=time (min)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

FIG. 7 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore(Lysenin—E76S/E84Q/E85K/E92Q/E97S/D126G/E167A, SEQ ID NO: 2 withmutations E76S/E84Q/E85K/E92Q/E97S/D126G/E167A) in a controlled fashion,producing stepwise changes in current as the DNA moves through thenanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time (s)) of a typical Hel308 Mbu controlled DNA movement (±180mV, 625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II),25 mM potassium ferricyanide(III), pH 8.0, 1 mM ATP, 10 mM MgCl₂, 0.6 nMDNA (SEQ ID NO: 13 and 14), 100 nM Hel308 Mbu (SEQ ID NO: 15),Lysenin-E76S/E84Q/E85K/E92Q/E97S/D126G/E167A, SEQ ID NO: 2 withmutations E76S/E84Q/E85K/E92Q/E97S/D126G/E167A)). B) Shows an expandedview of the helicase controlled DNA movement (y-axis=current (pA),x-axis=time (s)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

FIG. 8 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore(Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/E50S, SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S/D126G/E167A/E50S) in a controlled fashion,producing stepwise changes in current as the DNA moves through thenanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time (s)) of a typical Hel308 Mbu controlled DNA movement (+120mV, 625 mM KCl, 100 rnM Hepes pH 8.0, 75 mM potassium ferrocyanide(II),25 mM potassium ferricyanide(III), pH 8.0, 1 mM ATP, 10 mM MgCl₂, 0.3 nMDNA (SEQ ID NO: 13 and 14), 100 nM Hel308 Mbu (SEQ ID NO: 15),Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/E50S, SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S/D126G/E167A/E50S)). B) Shows an expandedview of the helicase controlled DNA movement (y-axis=current (pA),x-axis=time (s)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

FIG. 9 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore(Lysenin—E84Q/E85K/E92Q/E97S/D126G/E167A/E71S, SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S/D126(1/E167A/E71S) in a controlledfashion, producing stepwise changes in current as the DNA moves throughthe nanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time (Tran)) of a typical Hel308. Mbu controlled DNA movement(+180 mV, 625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassiumferrocyanide(II), 25 mM potassium ferricyanide(III), pH 8.0, 1 mM ATP,10 mM MgCl₂, 0.3 nM DNA (SEQ ID NO: 13 and 14), 100 nM Hel308 Mbu (SEQID NO: 15), Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/E71S, SEQ ID NO: 2with mutations E84Q/E85K/E92Q/E97S/D126G/E167A/E71S). B) Shows anexpanded view of the helicase controlled DNA movement (y-axis=current(pA), x-axis=time (s)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

FIG. 10 shows that Hel308 Mbu (SEQ ID) NO: 15) was able to move DNAthrough a lysenin nanopore(Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/E128S, SEQ NO: 2 with mutationsE84Q/E85K/E92Q/E97S/D126G/E167A/E128S) in a controlled fashion,producing stepwise changes in current as the DNA moves through thenanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time (s)) of a typical Hel308 Mbu controlled DNA movement (+180mV, 625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II),25 mM potassium ferricyanide(III), pH 8.0, 1 mM ATP, 10 mM MgCl₂, 0.6 nMDNA (SEQ ID NO: 13 and 14), 100 nM Hel308 Mbu (SEQ ID NO: 15),Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/E128S, SEQ ID) NO: 2 withmutations E84Q/E85K/E92Q/E97S/D126G/E167A/E128S). B) Shows an expandedview of the helicase controlled DNA movement (y-axis===current (pA),x-axis=time (s)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

FIG. 11 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore(Lysenin—E84Q/E85K/E92Q/E97S/D126G/E167A/D68S, SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S,T)126G/E167A/D68S) in a controlledfashion, producing stepwise changes in current as the DNA moves throughthe nanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time (min)) of a typical Hel308 Mbu controlled DNA movement (+120mV, 625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II),25 mM potassium ferricyanide(III), pH 8.0, 1 mM ATP, 10 mM MgCl₂, 0.3 nMDNA (SEQ ID NO: 13 and 14), 100 nM Hel308 Mbu (SEQ ID NO: 15),Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/D68S, SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S/D126G/E167A/D68S). B) Shows an expandedview of the helicase controlled DNA movement (y-axis=current (pA),x-axis=time (s)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

FIG. 12 shows that Hel308 Mbu (SEQ ID NO: 15) was able to move DNAthrough a lysenin nanopore(Lysenin—E84Q/E85K/E92Q/E97S/D126G/E167A/D121S, SEQ ID NO: 2 withmutations E840/E85K/E92Q/E97S/D126G/E167A/D121S) in a controlledfashion, producing stepwise changes in current as the DNA moves throughthe nanopore. A) Shows an example current trace (y-axis=current (pA),x-axis=time (s)) of a typical a Hel308 Mbu controlled DNA movement (+120mV, 625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II),25 mM potassium ferricyanide(III), pH 8.0, 1 mM ATP, 10 mM MgCl₂, 0.6 nMDNA (SEQ ID NO: 13 and 14), 100 nM Hel308 Mbu (SEQ ID NO: 15),Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A/D121S, SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S/D126G/E167A/D121S). B) Shows an expandedview of the helicase controlled DNA movement (y-axis=current (pA),x-axis=time (s)) in the upper trace. The DNA level shows stepwisechanges in current as the enzyme moves the DNA through the pore.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the polynucleotide sequence encoding the lyseninmonomer.

SEQ ID NO: 2 shows the amino acid sequence of the lysenin monomer.

SEQ ID NO: 3 shows the polynucleotide sequence encoding the Phi29 DNApolymerase,

SEQ ID NO: 4 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 5 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 6 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 7 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 8 shows the amino acid sequence of the exonuclease III enzymefrom E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 9 shows the codon optimised polynucleotide sequence derivedfrom the rec.I gene from T. thermophilus. It encodes the RecJ enzymefrom T. thermophilus (TthRecJ-cd).

SEQ ID NO: 10 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme per processive digestion of 5′monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 11 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 12 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′ direction(www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on astrand preferentially requires a 5′ overhang of approximately 4nucleotides with a 5′ phosphate.

SEQ ID NO: 13 and 14 show the polynucleotide sequences of ssDNA used inExamples 1, 2, 3, 4, 5 and 6. SEQ ID NO: 14 has a 3′-cholesterol tag.

SEQ ID NO: 15 shows the amino acid sequence of Hel308 Mbu.

SEQ ID NO: 16 shows the amino acid sequence of lysenin related protein(LRP) 1.

SEQ ID NO: 17 shows the amino acid sequence of lysenin related protein(LRP) 1.

SEQ ft) NO: 18 shows the amino acid sequence of lysenin related protein(LRP) 1.

SEQ ID NO: 19 shows the amino acid sequence of the activated version ofparasporin-2.

The full length protein is cleaved at its amino and carboxy termini toform an activated version that is capable of forming pores.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “amutant monomer” includes “mutant monomers”, reference to “asubstitution” includes two or more such substitutions, reference to “apore” includes two or more such pores, reference to “a polynucleotide”includes two or more such polynucleotides, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Mutant Lysenin Monomers

The present invention provides mutant lysenin monomers. The mutantlysenin monomers may be used to form the pores of the invention. Amutant lysenin monomer is a monomer whose sequence varies from that of awild-type lysenin monomer (i.e. SEQ ID NO: 2) and which retains theability to form a pore in the presence of other monomers of theinvention or other monomers from lysenin or derived from lysenin.Methods for confirming the ability of mutant monomers to form pores arewell-known in the art and are discussed in more detail below. Forinstance, the ability of a mutant monomer to form a pore can bedetermined as described in Example 1.

The mutant monomers have an altered ability to interact with apolynucleotide. Pores comprising one or more of the mutant monomerstherefore have improved nucleotide reading properties e.g. display (1)improved polynucleotide capture and (2) improved polynucleotiderecognition or discrimination. In particular, pores constructed from themutant monomers capture nucleotides and polynucleotides more easily thanthe wild type. In addition, pores constructed from the mutant monomersdisplay an increased current range, which makes it easier todiscriminate between different nucleotides, and a reduced variance ofstates, which increases the signal-to-noise ratio. In addition, thenumber of nucleotides contributing to the current as the polynucleotidemoves through pores constructed from the mutants is decreased. Thismakes it easier to identify a direct relationship between the observedcurrent as the polyaucleotide moves through the pore and thepolynucleotide. The improved nucleotide reading properties of themutants are achieved via five main mechanisms, namely by changes in the:

-   -   sterics (increasing or decreasing the size of amino acid        residues);    -   charge (e.g. introducing or removing −ve charge and/or        introducing or removing +ve charge);    -   hydrogen bonding (e.g. introducing amino acids that can hydrogen        bond to the base pairs);    -   pi stacking (e.g. introducing amino acids that interact through        delocalised elec pi systems); and/or    -   alteration of the structure of the pore (e.g. introducing amino        acids that increase the size of the barrel or channel).

Any one or more of these five mechanisms may be responsible for theimproved properties of the pores formed from the mutant monomers of theinvention. For instance, a pore comprising a mutant monomer of theinvention may display improved nucleotide reading properties as a resultof altered sterics, altered hydrogen bonding and an altered structure.

A mutant monomer of the invention comprises a variant of the sequenceshown in SEQ ID NO: 2. SEQ ID NO: 2 is the wild-type sequence of thelysenin monomer. A variant of SEQ ID NO: 2 is a polypeptide that has anamino acid sequence which varies from that of SEQ ID NO: 2 and whichretains its ability to form a pore.

The inventors have surprisingly identified a region within the lyseninmonomer which can be modified to alter the interaction between themonomer and a polynucleotide, such as when the polynucleotide ischaracterised using nanopore sensing with a pore comprising the monomer.The region is from about position 44 to about position 126 of SEQ ID NO:2. At least a part of this region typically contributes to the membranespanning region of lysenin. At least a part of this region typicallycontributes to the barrel or channel of lysenin. At least a part of thisregion typically contributes to the internal wall or lining of lysenin.

The transmembrane region of lysenin has been identified as positions 44to 67 of SEQ NO. 2 (De Colbis et al., Structure, 2012; 20: 1498-1507).

In accordance with the invention, the variant comprises one or moremodifications within the region of from about position 44 to aboutposition 126 of SEQ ID NO: 2 which alter the ability of the monomer, orpreferably the region, to interact with a polynucleotide. Theinteraction between the monomer and a polynucleotide may be increased ordecreased. An increased interaction between the monomer and apolynucleotide will, for example, facilitate capture of thepolynucleotide by pores comprising the mutant monomer. A decreasedinteraction between the region and a polynucleotide will, for example,improve recognition or discrimination of the polynucleotide. Recognitionor discrimination of the polynucleotide may be improved by decreasingthe variance of states of pores comprising the mutant monomer (whichincreases the signal-to-noise ratio) and/or decreasing the number ofnucleotides in the polynucleotide contributing to the current as thepolynucleotide moves through pores comprising the mutant monomer.

The invention therefore provides a mutant lysenin monomer comprising avariant of the sequence shown in SEQ ID NO: 2, wherein the monomer iscapable of forming a pore and wherein the variant comprises one or moremodifications from about position 44 to about position 126 of SEQ ID NO:2 which alter the ability of the monomer to interact with apolynucleotide.

The ability of the monomer to interact with a polynucleotide can bedetermined using methods that are well-known in the art. The monomer mayinteract with a polynucleotide in any way, e.g. by non-covalentinteractions, such as hydrophobic interactions, hydrogen bonding, Vander Waal's forces, pi (π)-cation interactions or electrostatic forces.For instance, the ability of the region to bind to a polynucleotide canbe measured using a conventional binding assay. Suitable assays include,but are not limited to, fluorescence-based binding assays, nuclearmagnetic resonance (NMR), Isothermal Titration calorimetry (ITC) orElectron spin resonance (ESR) spectroscopy. Alternatively, the abilityof a pore comprising one or more of the mutant monomers to interact witha polynucleotide can be determined using any of the methods discussedabove or below. Preferred assays are described in the Examples.

The one or more modifications are within the region from about position44 to about position 126 of SEQ ID NO: 2. The one or more modificationsare preferably within any one of the following regions: from aboutposition 40 to about position 125, from about position 50 to aboutposition 120, from about position 60 to about position 110 and fromabout position 70 to about position 100. IT the one or moremodifications are being made to improve polynucleotide capture, they aremore preferably made within any one of the following regions: from aboutposition 44 to about position 103, from about position 68 to aboutposition 103, from about position 84 to about position 103, from aboutposition 44 to about position 97, from about position 68 to aboutposition 97 or from about position 84 to about position 97. If the oneor more modifications are being made to improve polynucleotiderecognition or discrimination, they are more preferably made within anyone of the following regions: from about position 44 to about position109, from about position 44 to about position 97 or from about position48 to about position 88. The region is preferably from about position 44to about position 67 of SEQ ID NO: 2.

If the one or more modifications are intended improve polynucleotiderecognition or discrimination, they are preferably made in addition toone or more modifications to improve polynucleotide capture. This allowspores formed from the mutant monomer to effectively capture apolynucleotide and then characterise the polynucleotide, such asestimate its sequence, as discussed below.

Modifications of protein nanopores that alter their ability to interactwith a polynucleotide, in particular improve their ability to captureand/or recognise or discriminate polynucleotides, are well documented inthe art. For instance, such modifications are disclosed in WO2010/034018 and WO 2010/055307. Similar modifications can be made to thelysenin monomer in accordance with this invention.

Any number of modifications may be made, such as 1, 2, 5, 10, 15, 20, 30or more modifications. Any modification(s) can be made as long as theability of the monomer to interact with a polynucleotide is altered.Suitable modifications include, but are not limited to, amino acidsubstitutions, amino acid additions and amino acid deletions. The one ormore modifications are preferably one or more substitutions. This isdiscussed in more detail below.

The one or more modifications preferably (a) alter the steric effect ofthe monomer, or preferably alter the steric effect of the region, (b)alter the net charge of the monomer, or preferably alter the net chargeof the region, (c) alter the ability of the monomer, or preferably ofthe region, to hydrogen bond with the polynucleotide, (d) introduce orremove chemical groups that interact through delocalized electron pisystems and/or (e) alter the structure of the monomer, or preferablyalter the structure of the region. The one or more modifications morepreferably result in any combination of (a) to (e), such as (a) and (b);(a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and(e), (c) and (d); (c) and (e); (d) and (e), (a), (b) and (c); (a), (b)and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (e); (a), (d)and (e); (b), (c) and (d); (b), (c) and (e); (b), (d) and (e); (c), (d)and (e); (a), (b), (c) and d), (a), (b), (c) and (e), (a), (b), (d) and(e); (a), (c), (d) and (e); (b), (c), (d) and (e); and (a), (b), (c) and(d).

For (a), the steric effect of the monomer can be increased or decreased.Any method of altering the steric effects may be used in accordance withthe invention. The introduction of bulky residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H), increasesthe sterics of the monomer. The one or more modifications are preferablythe introduction of one or more of F, W, Y and H. Any combination of F,W, Y and H may be introduced. The one or more of F, W, Y and H may beintroduced by addition. The one or more of F, W, Y and H are preferablyintroduced by substitution. Suitable positions for the introduction ofsuch residues are discussed in more detail below.

The removal of bulky residues, such as phenylalanine (F), tryptophan(W), tyrosine (Y) or histidine (H), conversely decreases the sterics ofthe monomer. The one or more modifications are preferably the removal ofone or more of F, W, Y and H. Any combination of F, W, Y and H may beremoved. The one or more of F, W, Y and H may be removed by deletion.The one or more of F, W, Y and H are preferably removed by substitutionwith residues having smaller side groups, such as serine (S), threonine(I), alanine (A) and valine (V).

For (b), the net charge can be altered in any way. The net positivecharge is preferably increased or decreased. The net positive charge canbe increased in any manner. The net positive charge is preferablyincreased by introducing, preferably by substitution, one or morepositively charged amino acids and/or neutralising, preferably bysubstitution, one or more negative charges.

The net positive charge is preferably increased by introducing one ormore positively charged amino acids. The one or more positively chargedamino acids may be introduced by addition. The one or more positivelycharged amino acids are preferably introduced by substitution. Apositively charged amino acid is an amino acid with a net positivecharge. The positively charged amino acid(s) can be naturally-occurringor non-naturally-occurring. The positively charged amino acids may besynthetic or modified. For instance, modified amino acids with a netpositive charge may be specifically designed for use in the invention. Anumber of different types of modification to amino acids are well knownin the art.

Preferred naturally-occurring positively charged amino acids include,but are not limited to, histidine (H), lysine (K) and arginine (R). Theone or more modifications are preferably the introduction of one or moreof H, K and R. Any number and combination of H, K and R may beintroduced. The one or more of H, K and R may be introduced by addition.The one or more of H, K and R are preferably introduced by substitution.Suitable positions for the introduction of such residues are discussedin more detail below.

Methods for adding or substituting naturally-occurring amino acids arewell known in the art. For instance, methionine (M) may be substitutedwith arginine (R) by replacing the codon for methionine (ATG) with acodon for arginine (AGA) at the relevant position in a polynucleotideencoding the monomer. The polynucleotide can then be expressed asdiscussed below.

Methods for adding or substituting non-naturally-occurring amino acidsare also well known in the art. For instance, non-naturally-occurringamino acids may be introduced by including synthetic. aminoacyl-tRNAs inthe IVTT system used to express the pore. Alternatively, they may beintroduced by expressing the monomer in E. coli that are auxotrophic forspecific amino acids in the presence of synthetic (i.e.non-naturally-occurring) analogues of those specific amino acids. Theymay also be produced by naked ligation if the pore is produced usingpartial peptide synthesis.

Any amino acid may be substituted with a positively charged amino acid.One or more uncharged amino acids, non-polar amino acids and/or aromaticamino acids may be substituted with one or more positively charged aminoacids. Uncharged amino acids have no net charge. Suitable unchargedamino acids include, but are not limited to, cysteine (C), serine (5),threonine (T), methionine (M), asparagine (N) and glutamine (Q).Non-polar amino acids have non-polar side chains. Suitable non-polaramino acids include, but are not limited to, glycine (G), alanine (A),proline (P), isoleucine (I), leucine (L) and valine (V). Aromatic aminoacids have an aromatic side chain. Suitable aromatic amino acidsinclude, but are not limited to, histidine (H), phenylalanine (F),tryptophan (W) and tyrosine (Y). Preferably, one or more negativelycharged amino acids are substituted with one or more positively chargedamino acids. Suitable negatively charged amino acids include, but arenot limited to, aspartic acid (D) and glutamic acid (E).

Preferred introductions include, but are not limited to, substitution ofsubstitution E with K, M with R, substitution of M with H, substitutionof M with K, substitution of D with R, substitution of D with H,substitution of D with K, substitution of E with R, substitution of Ewith H, substitution of N with R, substitution of T with R andsubstitution of G with R. Most preferably E is substituted with K.

Any number of positively charged amino acids may be introduced orsubstituted. For instance, 1, 2, 5, 10, 15, 20, 25, 30 or morepositively charged amino acids may be introduced or substituted.

The net positive charge is more preferably increased by neutralising oneor more negative charges. The one or more negative charges may beneutralised by replacing by substitution one or more negatively chargedamino acids with one or more uncharged amino acids, non-polar aminoacids and/or aromatic amino acids. The removal of negative chargeincreases the net positive charge. The uncharged amino acids, non-polaramino acids and/or aromatic amino acids can be naturally-occurring ornon-naturally-occurring. They may be synthetic or modified. Suitableuncharged amino acids, non-polar amino acids and aromatic amino acidsare discussed above. Preferred substitutions include, but are notlimited to, substitution of E with Q, substitution of E with S,substitution of F with A, substitution of D with Q, substitution of Ewith N, substitution of D with N, substitution of D with G andsubstitution of D with S.

Any number and combination of uncharged amino acids, non-polar aminoacids and/or aromatic amino acids may substituted. For instance, 1, 2,5, 10, 15, 20, 25, or 30 or more uncharged amino acids, non-polar aminoacids and/or aromatic amino acids may be substituted. Negatively chargedamino acids may be substituted with (1) uncharged amino acids; (2)non-polar amino acids; (3) aromatic amino acids; (4) uncharged aminoacids and non-polar amino acids; (5) uncharged amino acids and aromaticamino acids; and (5) non-polar amino acids and aromatic amino acids; or(6) uncharged amino acids, non-polar amino acids and aromatic aminoacids.

The one or more negative charges may be neutralised by introducing oneor more positively charged amino acids near to, such as within 1, 2, 3or 4 amino acids, or adjacent to one or more negatively charged aminoacids. Examples of positively and negatively charged amino acids arediscussed above. The positively charged amino acids may be introduced inany manner discussed above, for instance by substitution.

The net positive charge is preferably decreased by introducing one ormore negatively charged amino acids and/or neutralising one or morepositive charges. Ways in which this might be done will be clear fromthe discussion above with reference to increasing the net positivecharge. All of the embodiments discussed above with reference toincreasing the net positive charge equally apply to decreasing the netpositive charge except the charge is altered in the opposite way. Inparticular, the one or more positive charges are preferably neutralisedby substituting one or more positively charged amino acids with one ormore uncharged amino acids, non-polar amino acids and/or aromatic aminoacids or by introducing one or more negatively charged amino acids nearto, such as within 1, 3 or 4 amino acids of, or adjacent to one or morenegatively charged amino acids.

The net negative charge is preferably increased or decreased. All of theabove embodiments discussed above with reference to increasing ordecreasing the net positive charge equally apply to decreasing orincreasing the net negative charge respectively.

For (c), the ability of the monomer to hydrogen bond may be altered inany manner. The introduction of serine (S), threonine (I), asparagine(N), glutamine (Q), tyrosine (Y) or histidine (H) increases the hydrogenbonding ability of the monomer. The one or more modifications arepreferably the introduction of one or more of S, T, N, Q, Y and H. Anycombination of S, T, N, Q, Y and H may be introduced. The one or more ofS, T, N, Q, Y and H may be introduced by addition. The one or more of S,T, N, Q, Y and H are preferably introduced by substitution. Suitablepositions for the introduction of such residues are discussed in moredetail below.

The removal of serine (S), threonine (T), asparagine (N), glutamine (Q),tyrosine (Y) or histidine (H) decreases the hydrogen bonding ability ofthe monomer. The one or more modifications are preferably the removal ofone or more of S, T, N, Q, Y and H. Any combination of S, T, N, Q, Y andH may be removed. The one or more of S, T, N, Q, Y and H may be removedby deletion. The one or more of S, T, N, Q, Y and H are preferablyremoved by substitution with other amino acids which hydrogen bond lesswell, such as alanine (A), valine (V), isoleucine (I) and leucine (L).

For (d), the introduction of aromatic residues, such as phenylalanine(F), tryptophan (W), tyrosine (Y) or histidine (H), also increases thepi stacking in the monomer. The removal of aromatic residues, such asphenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H), alsoincreases the pi stacking in the monomer. Such amino acids can beintroduced or removed as discussed above with reference to (a).

For (e), one or more modifications made in accordance with the inventionwhich alter the structure of the monomer. For example, one or More loopregions can be removed, shortened or extended. This typicallyfacilitates the entry or exit of a polynucleotide into or out of thepore. The one or more loop regions may be the cis side of the pore, thetrans side of the pore or on both sides of the pore. Alternatively, oneor more regions of the amino terminus and/or the carboxy terminus of thepore can be extended or deleted. This typically alters the size and/orcharge of the pore.

It will be clear from the discussion above that the introduction ofcertain amino acids will enhance the ability of the monomer to interactwith a polynucleotide via more than one mechanism. For instance, thesubstitution of E with H will not only increase the net positive charge(by neutralising negative charge) in accordance with (b), but will alsoincrease the ability of the monomer to hydrogen bond in accordance with(c).

The variant preferably comprises a substitution at one or more of thefollowing positions of SEQ ID NO: 2: M44, N46, N48, E50, R52, H58, D68,F70, E71, S74, E76, S78, Y79, S80, H81, S82, E84, E85, S86, Q87, S89,M90, E92, E94, E97, E102, H103, T104, T106, R115, Q117, N119, D121 andD126. The variant preferably comprises a substitution at 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 of those positions. The variantpreferably comprises a substitution at one or more of the followingpositions of SEQ ID NO: 2: D68, E71, S74, E76, S78, S80, S82, E84, E85,S86, Q87, S89, E92, E102, T104, T106, R115, Q117, N119 and D121. Thevariant preferably comprises a substitution at 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of those positions. Theamino acids substituted into the variant may be naturally-occurring ornon-naturally occurring derivatives thereof. The amino acids substitutedinto the variant may be D-amino acids. Each position listed above may besubstituted with asparagine (N), serine (S), glutamine (Q), arginine(R), glycine (G), tyrosine (Y), aspartic acid (D), leucine (L), lysine(K) or alanine (A).

The variant preferably comprises at last one of the following mutationsof SEQ 1D NO: 2:

(a) serine (S) at position 44;

(b) serine (S) at position 46;

(c) serine (S) at position 48;

(d) serine (S) at position 52;

(e) serine (S) at position 58;

(f) serine (S) at position 68;

(g) serine (S) at position 70;

(h) serine (S) at position 71;

(i) serine (S) at position 76;

(j) serine (S) at position 79;

(k) serine (S) at position 81;

(l) serine (S), aspartic acid (D) or glutamine at position 84;

(m) serine (S) or lysine (K) at position 85;

(n) serine (S) at position 87;

(o) serine (5) at position 90;

(p) asparagine (N) or glutamine (Q) at position 92;

(q) serine (S) or asparagine (N) at position 94;

(r) serine (S) or asparagine (N) at position 97;

(s) serine (S) at position 102;

serine (S) at position 103;

(u) asparagine (N) or serine (S) at position 121;

(v) serine (S) at position 50;

(w) asparagine (N) or serine (S) at position 94;

(x) asparagine (N) or serine (S) at position 97;

(y) serine (S) or asparagine (N) at position 121;

(z) asparagine (N) or glutamine (Q) or glycine at position 126; and

(aa) serine (S) or asparagine (N) at position 128.

The variant may include any number of mutations (a) to (aa), such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26 or 27 of the mutations. Preferred combinations ofmutations are discussed below. The amino acids introduced into thevariant may be naturally-occurring or non-naturally occurringderivatives thereof. The amino acids introduced into the variant may beD-amino acids.

The variant preferably comprises at least one of the following mutationsof SEQ It) NO: 2:

(a) serine (S) at position 68;

(b) serine (S) at position 71;

(c) serine (S) at position 76;

(d) aspartic acid (D) or glutamine (Q) at position 84;

(e) lysine (K) at position 85;

(f) asparagine (N) or glutamine (Q) at position 92;

(g) serine (S) at position 102;

(h) asparagine (N) or serine (S) at position 121;

(i) serine (S) at position 50;

(j) asparagine (N) or serine (S) at position 94;

(k) asparagine (N) or serine (S) at position 97; and

(l) asparagine (N) or glutamine (Q) or glycine (G) at position 126.

The variant may include any number of mutations (a) to (1), such as 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the mutations. Preferredcombinations of mutations are discussed below. The amino acidsintroduced into the variant may be naturally-occurring or non-naturallyoccurring derivatives thereof. The amino acids introduced into thevariant may be D-amino acids.

The variant may include one or more additional modifications outside ofthe region of from about position 44 to about position 126 of SEQ ID NO:2 which in combination with the modifications in the region discussedabove improve polynucleotide capture and/or improve polynucleotiderecognition or discrimination. Suitable modifications include, but arenot limited to, substitution at one or more of D35, E128, E135, E134 andE167. In particular, removal of the negative charge by substituting Eatone or more of positions 128, 135, 134 and 167 improves polynucleotidecapture. E at one or more of these positions may be substituted in anyof the ways discussed above. Preferably all of E128, E135, E134 and E167are substituted as discussed above. E is preferably substituted with A.In other words, the variant preferably comprises one or more of, or allof, E128A, E135A, E134A and E167A. Another preferred substitution isD3.5Q.

In a preferred embodiment, the variant comprises the followingsubstitutions in SEQ ID NO: 2:

-   -   i. one or more of, such as both of, E84D and E85K;    -   ii. one or more of, such as 2, 3, 4, 5 or 6 of, E84Q, E85K,        E92Q, E97S, D126G and E167A;    -   iii. one or more of, such as 2, 3, 4 or 5 of, E92N, E94N, E97N,        D121N and D126N;    -   iv. one or more of, such as 2, 3, 4, 5 or 6 of, E92N, E94N,        E97N, D121N, D126N and E128N;    -   v. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E76S, E84Q,        E85K, E92Q, E97S, D126G and E167A;    -   vi. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and E50S;    -   vii. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and E71S;    -   viii. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and E94S;    -   ix. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and E102S;    -   x. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and E128S;    -   xi. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and E135S;    -   xii. one or more of, such as 2, 3, 4, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and D68S;    -   xiii. one or more of, such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and D121S;    -   xiv. one or more of such as 2, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q, E97S, D126G, E167A and D134S;    -   xv. one or more of, such as 2 or 3 of, E84D, E85K and E92Q;    -   xvi. one or more of, such as 1, 2, 3, 4, 5 or 6 of, E84Q, E85K,        E92Q, E97S, D126G and E135S;    -   xvii. one or more of, such as 1, 2, 3, 4 or 5 of, E85K, E92Q,        E94S, E97S and D126G,    -   xviii. one or more of, such as 1, 2, 3, 4 or 5 of, E76S, E85K,        E92Q, E97S and D126G;    -   xix. one or more of, such as 1, 2, 3, 4 or 5 of, E71S, E85K,        E92Q, E97S and D126G;    -   xx. one or more of, such as 1, 2, 3, 4 or 5 of, D68S, E85K,        E92Q, E97S and D126G;    -   xxi. one or more of, such as 1, 2, 3 or 4 of, E85K, E92Q, E97S        and D126G;    -   xxii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, E84Q, E85K,        E92Q, E97S, H103S and D126G;    -   xxiii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, E84Q,        E85K, M90S, E92Q, E97S and D126G;    -   xxiv. one or more of, such as 1, 2, 3, 4, 5 or 6 of, E84Q, Q87S,        E85K, E92Q, E97S and D126G;    -   xxv. one or more of, such as 1, 2, 3, 4 or 5 of, E84Q, E85S,        E92Q, E97S and D126G;    -   xxvi. one or more of, such as 1, 2, 3, 4 or 5 of, E84S, E85K,        E92Q, E97S and D126G;    -   xxvii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, H81S,        E84Q, E85K, E92Q, E97S and D126G;    -   xxviii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, Y79S,        E84Q, E85K, E92Q, E97S and D126G;    -   xxix. one or more of, such as 1, 2, 3, 4, 5 or 6 of, F70S, E84Q,        E85K, E92Q, E97S and D126G,    -   xxx. one or more of, such as 1, 2, 3, 4, 5 or 6 of, H58S, E84Q,        E85K, E92Q, E97S and D126G,    -   xxxi. one or more of, such as 1, 2, 3, 4, 5 or 6 of, R52S, E84Q,        E85K, E92Q, E97S and D126G;    -   xxxii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, N48S,        E84Q, E85K, E92Q, E97S and D126G;    -   xxxiii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, N46S,        E84Q, E85K, E92Q, E97S and D126G;    -   xxxiv. one or more of, such as 1, 2, 3, 4, 5 or 6 of, M44S,        E84Q, E85K, E92Q, E97S and D126G;    -   xxxv. one or more of, such as both of, E92Q and E97S;    -   xxxvi. one or more of, such as 1, 2, 3 or 4 of, ES84Q, E85K,        E92Q and E97S;    -   xxxvii. one or more of, such as both of, E84Q and E85K;    -   xxxviii. one or more of, such as 1, 2 or 3 of, E84Q, E85K and        D126G;    -   xxxix. one or more of, such as 1, 2, 3 or 4 of, E84Q, E85K,        D126G and E167A;    -   xl. one or more of, such as 1, 2 or 3 of, E92Q, E97S and D126G;    -   xli. one or more of, such as 1, 2, 3, 4 or 5 of, E84Q, E85K,        E92Q, E97S and D126G;    -   xlii. one or more of, such as 1, 2, 3, 4 or 5 of, E84Q, E85K,        E92Q, E97S and E167A;    -   xliii. one or more of, such as 1, 2, 3, 4 or 5 of, E84Q, E85K,        E92Q, D126G and E167A;    -   xliv. one or more of, such as 1, 2, 3, 4 or 5 of, E84Q, E85K,        E97S, D126G and E167A,    -   xlv. one or more of, such as 1, 2, 3, 4 or 5 of, ES4Q, E92Q,        E97S, D126G and E167A;    -   xlvi. one or more of, such as 1, 2, 3, 4 or 5 of, E85K, E92Q,        E97S, D126G and E167A;    -   xlvii. one or more of, such as 1, 2 or 3 of, E84D, E85K and        E92Q;    -   xlviii. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q, E97S, D126G, E167A and D121S;    -   xlix. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q, E97S, D126G, E167A and D68S;    -   l. one or more of, such as 1, 3, 4, 5, 6 or 7 of, E84Q, E85K,        E92Q E97S, D126G, E167A and E135S;    -   li. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q E97S, D126G, E167A and E128S;    -   lii. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q, E97S, D126G, E167A and E102S;    -   liii. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q, E97S, D126G, E167A and E94S;    -   liv. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q, E97S, D126G, E167A and E71S;    -   lv. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E84Q,        E85K, E92Q, E97S, D126G, E167A and E50S;    -   lvi. one or more of, such as 1, 2, 3, 4, 5, 6 or 7 of, E76S,        E84Q, E85K, E92Q, E97S, D126G and E167A;    -   lvii. one or more of, such as 1, 2, 3, 4, 5 or 6 of, E92N, E94N,        E97N, D121N, D126N and E128N;    -   lviii. one or more of, such as 1, 2, 3, 4 or 5 of E92N, E94N,        E97N, D121N and D126N; or    -   lix. one or more of, such as 1, 2, 3, 4, 5 or 6 of, E84Q, E85K,        E92Q, E97S, D126G and E167A

In the above, the first letter refers to the amino acid in SEQ ID NO: 2being replaced, the number is the position in SEQ ID NO: 2 and thesecond letter refers to the amino acid with which the first is besubstituted. Hence, E84D refers to substitution of glutamic acid (E) atposition 84 with aspartic acid (D).

The variant may include any number of the substitutions in any one of ito lix, such as 1, 2, 3, 4, 5, 6 or 7. The variant preferably includesall of the substitutions shown in any one of i to lix above.

In a preferred embodiment, the variant comprises the substitutions inany one of i to xv above. The variant may include any number of thesubstitutions in any one of i to xv, such as 1, 2, 3, 4, 5, 6 or 7. Thevariant preferably includes all of the substitutions shown in any one ofi to xv above.

If the one or more modifications are intended to improve the ability ofthe monomer to recognise or discriminate a polynucleotide, they arepreferably made in addition to the modifications discussed above thatimprove polynucleotide capture, such as E84Q,1E85K, E92Q, E97S, D126Gand E167A.

The one or more modifications made to the identified region may concernthe substitution of one or more amino acids in the region with aminoacids present at the corresponding position(s) in homologues orparalogues of lysenin. Four examples of homologues of lysenin are shownin SEQ ID NOs: 16 to 19. The advantage of such substitutions is thatthey are likely to result in mutant monomers that form pores since thehomologue monomers also form pores.

In addition to the specific mutations discussed above, the variant mayinclude other mutations. These mutations do not necessarily enhance theability of the monomer to interact with a polynucleotide. The mutationsmay facilitate, for example, expression and/or purification. Over theentire length of the amino acid sequence of SEQ ID NO: 2, a variant willpreferably be at least 50% homologous to that sequence based on aminoacid identity. More preferably, the variant may be at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90% and more preferably at least 95%, 97% or 99%homologous based on amino acid identity to the amino acid sequence ofSEQ ID NO: 2 over the entire sequence. There may be at least 80%, forexample at least 85%, 90% or 95%, amino acid identity over a stretch of100 or more, for example 125, 150, 175 or 200 or more, contiguous aminoacids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pair(HSPs) by identifying short words of length W in the query sequence thateither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighbourhood word score threshold (Altschul et al,supra). These initial neighbourhood word hits act as seeds forinitiating searches to find HSP's containing them. The word hits areextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extensions for the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a word length (W) of 11, BLOSUM62 scoring matrix (see Henikoffand Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two amino acidsequences would occur by chance. For example, a sequence is consideredsimilar to another sequence if the smallest sum probability incomparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 1.below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 2.

TABLE 1 Chemical properties of amino acids Ala aliphatic, hydrophobic,neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asnpolar, hydrophilic, neutral Asp polar, hydrophilic, charged (−) Prohydrophobic, neutral Glu polar, hydrophilic, charged (−) Gln polar,hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar,hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, hydrophilic, charged (+) Thr polar,hydrophilic, neutral Ile aliphatic, hydrophobic, neutral Val aliphatic,hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trp aromatic,hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic,polar, hydrophobic

TABLE 2 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

The variant may comprise one or more substitutions outside of the regionspecified above in which amino acids are replaced with those at thecorresponding position(s) in homologues and paralogues of lysenin. Fourexamples of homologues of lysenin are shown in SEQ NOs: 16 to 19.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the variants described above. Up to1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. This may be assayed as described above. Fragmentsmay be at least 50, 100, 150, 200 or 250 amino acids in length. Suchfragments may be used to produce the pores of the invention. Since theregion of from about position 44 to about position 126 of SEQ ID NO: 2can be modified by one or more deletions in accordance with theinvention, a fragment does not have to contain the entire region. Hence,fragments shorter than the length of the unmodified region are envisagedby the invention. A fragment preferably comprises the pore formingdomain of SEQ ID NO: 2. A fragment more preferably comprises the regionfrom about position 44 to about position 126 of SEQ ID NO: 2 which ismodified in accordance with the invention.

One or more amino acids may be alternatively or additionally added tothe variants described above. An extension may be provided at the aminoterminal or carboxy terminal of the amino acid sequence of the variantof SEQ ID NO: 2, including a fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the region of SEQID NO: 2 that is responsible for pore formation, namely from aboutposition 44 to about position 126 and this region is modified inaccordance with the invention as discussed above. It may contain afragment of this region as discussed above. In addition to themodifications of the invention, a variant of SEQ ID NO: 2 may includeone or more additional modifications, such as substitutions, additionsor deletions. These modifications are preferably located in thestretches in the variant that correspond to from about position 1 toabout position 43 and from about position 127 to about position 297 ofSEQ ID NO: 2 (i.e. outside of the region modified in accordance with theinvention).

The mutant monomers may be modified to assist their identification orpurification, for example by the addition of histidine residues (a histtag), aspartic acid residues (an asp tag), a streptavidin tag or a flagtag, or by the addition of a signal sequence to promote their secretionfrom a cell where the polypeptide does not naturally contain such asequence. An alternative to introducing a genetic tag is to chemicallyreact a tag onto a native or engineered position on the pore. An exampleof this would be to react a gel-shift reagent to a cysteine engineeredon the outside of the pore. This has been demonstrated as a method forseparating hemolysin hetero-oligomers (Chem Biol. 1997 July;4(7):497-505).

The mutant monomer may be labelled with a revealing label. The revealinglabel may be any suitable label which allows the pore to be detected.Suitable labels include, but are not limited to, fluorescent molecules,radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides, polyethylene glycols (PEGs), peptides and ligands suchas biotin.

The mutant monomer may also be produced using D-amino acids. Forinstance, the mutant monomer may comprise a mixture of L-amino acids andD-amino acids. This is conventional in the art for producing suchproteins or peptides.

The mutant monomer contains one or more specific modifications tofacilitate interaction with a polynucleotide. The mutant monomer mayalso contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe mutant monomer. Such modifications include, for example, reductivealkylation of amino acids by reaction with an aldehyde followed byreduction with NaBH₄, amidination with methylacetimidate or acylationwith acetic anhydride.

The mutant monomer can be produced using standard methods known in theart. The monomer may be made synthetically or by recombinant means. Forexample, the monomer may be synthesized by in vitro translation andtranscription (IVTT). Suitable methods for producing pore monomers arediscussed in International Application Nos. PCT/GB09/001690 (publishedas WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603). Methods for insertingpores into membranes are discussed below.

Polynucleotide sequences encoding a mutant monomer may be derived andreplicated using standard methods in the art. Such sequences arediscussed in more detail below. Polynucleotide sequences encoding amutant monomer may be expressed in a bacterial host cell using standardtechniques in the art. The mutant monomer may be produced in a cell byin situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide.

A mutant monomer may be produced in large scale following purificationby any protein liquid chromatography system from pore producingorganisms or after recombinant expression as described below. Typicalprotein liquid chromatography systems include FPLC, AKTA systems, theBio-Cad system, the Bio-Rad Biologic system and the Gilson IIPLC system.The mutant monomer may then be inserted into a naturally occurring orartificial membrane for use in accordance with the invention. Methodsfor inserting pore into membranes are discussed below.

In some embodiments, the mutant monomer is chemically modified. Themutant monomer can be chemically modified in any way and at any site.The mutant monomer is preferably chemically modified by attachment of amolecule to one or more cysteines (cysteine linkage), attachment of amolecule to one or more lysines, attachment of a molecule to one or morenon-natural amino acids, enzyme modification of an epitope ormodification of a terminus. Suitable methods for carrying out suchmodifications are well-known in the art. Suitable non-natural aminoacids include, but are not limited to, 4-azido-L-phenylalanine (Faz) andany one of the amino acids numbered 1-71 in FIG. 1 of Liu C. C. andSchultz P. G. Annu. Rev. Biochem., 2010, 79, 413-444. The mutant monomermay be chemically modified by the attachment of any molecule. Forinstance, the mutant monomer may be chemically modified by attachment ofa polyethylene glycol (PEG), a nucleic acid, such as DNA, a dye, afluorophore or a chromophore.

In some embodiments, the mutant monomer is chemically modified with amolecular adaptor that facilitates the interaction between a porecomprising the monomer and a target analyte, a target nucleotide ortarget polynucleotide. The presence of the adaptor improves thehost-guest chemistry of the pore and the nucleotide or polynucleotideand thereby improves the sequencing ability of pores formed from themutant monomer. The principles of host-guest chemistry are well-known inthe art. The adaptor has an effect on the physical or chemicalproperties of the pore that improves its interaction with the nucleotideor polynucleotide. The adaptor may alter the charge of the barrel orchannel of the pore or specifically interact with or bind to thenucleotide or polynucleotide thereby facilitating its interaction withthe pore.

The molecular adaptor is preferably a cyclic molecule, for example acyclodextrin, a species that is capable of hybridization, a DNA binderor interchelator, a peptide or peptide analogue, a synthetic polymer, anaromatic planar molecule, a small positively-charged molecule or a smallmolecule capable of hydrogen-bonding.

The adaptor may be cyclic. A cyclic adaptor preferably has the samesymmetry as the pore.

The adaptor typically interacts with the analyte, nucleotide orpolynucleotide via host-guest chemistry. The adaptor is typicallycapable of interacting with the nucleotide or polynucleotide. Theadaptor comprises one or more chemical groups that are capable ofinteracting with the nucleotide or polynucleotide. The one or morechemical groups preferably interact with the nucleotide orpolynucleotide by non-covalent interactions, such as hydrophobicinteractions hydrogen bonding, Van der Waal's forces, π-cationinteractions and/or electrostatic forces. The one or more chemicalgroups that are capable of interacting with the nucleotide orpolynucleotide are preferably positively charged. The one or morechemical groups that are capable of interacting with the nucleotide orpolynucleotide more preferably comprise amino groups. The amino groupscan be attached to primary, secondary or tertiary carbon atoms. Theadaptor even more preferably comprises a ring of amino groups, such as aring of 6, 7, 8 or 9 amino groups. The adaptor most preferably comprisesa ring of 6 or 9 amino groups. A ring of protonated amino groups mayinteract with negatively charged phosphate groups in the nucleotide orpolynucleotide.

The correct positioning of the adaptor within the pore can befacilitated by host-guest chemistry between the adaptor and the porecomprising the mutant monomer. The adaptor preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore. The adaptor more preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore via non-covalent interactions, such ashydrophobic interactions, hydrogen bonding, Van der Waal's forces,π-cation interactions and/or electrostatic forces. The chemical groupsthat are capable of interacting with one or more amino acids in the poreare typically hydroxyls or amines. The hydroxyl groups can be attachedto primary, secondary or tertiary carbon atoms. The hydroxyl groups mayform hydrogen bonds with uncharged amino acids in the pore. Any adaptorthat facilitates the interaction between the pore and the nucleotide orpolynucleotide can be used.

Suitable adaptors include, but are not limited to, cyclodextrins, cyclicpeptides and cucurbiturils. The adaptor is preferably a cyclodextrin ora derivative thereof. The cyclodextrin or derivative thereof may be anyof those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The adaptor is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). The guanidinogroup in gu₇-βCD has a much higher pKa than the primary amines inam₇-βCD and so it more positively charged. This gu₇-βCD adaptor may beused to increase the dwell time of the nucleotide in the pore, toincrease the accuracy of the residual current measured, as well as toincrease the base detection rate at high temperatures or low dataacquisition rates.

If a succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker isused as discussed in more detail below, the adaptor is preferablyheptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl-β-cyclodextrin(am₆amPDP₁-βCD).

More suitable adaptors include γ-cyclodextrins, which comprise 8 sugarunits (and therefore have eight-fold symmetry). The γ-cyclodextrin maycontain a linker molecule or may be modified to comprise all or more ofthe modified sugar units used in the β-cyclodextrin examples discussedabove.

The molecular adaptor is preferably covalently attached to the mutantmonomer. The adaptor can be covalently attached to the pore using anymethod known in the art. The adaptor is typically attached via chemicallinkage. If the molecular adaptor is attached via cysteine linkage, theone or more cysteines have preferably been introduced to the mutant bysubstitution. The mutant monomers of the invention can of coursecomprise a cysteine residue at one or both of positions 272 and 283. Themutant monomer may be chemically modified by attachment of a molecularadaptor to one or both of these cysteines. Alternatively, the mutantmonomer may be chemically modified by attachment of a molecule to one ormore cysteines or non-natural amino acids, such as FAz, introduced atother positions.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the mutant monomer before a linker is attached. The moleculemay be attached directly to the mutant monomer. The molecule ispreferably attached to the mutant monomer using a linker, such as achemical crosslinker or a peptide linker.

Suitable chemical crosslinkers are well-known in the art. Preferredcrosslinkers include 2,5-dioxopyrrolidin-1-yl3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl8-(pyridin-2-yldisulfanyl)octananoate. The most preferred crosslinker issuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, themolecule is covalently attached to the bifunctional crosslinker beforethe molecule/crosslinker complex is covalently attached to the mutantmonomer but it is also possible to covalently attach the bifunctionalcrosslinker to the monomer before the bifunctional crosslinker/monomercomplex is attached to the molecule.

The linker is preferably resistant to dithiothreitol (DTT). Suitablelinkers include, but are not limited to, iodoacetamide-based andMaleimide-based linkers.

In other embodiment, the monomer may be attached to a polynucleotidebinding protein. This forms a modular sequencing system that may be usedin the methods of the invention. Polynucleotide binding proteins arediscussed below.

The polynucleotide binding protein may be covalently attached to themutant monomer. The protein can be covalently attached to the pore usingany method known in the art. The monomer and protein may be chemicallyfused or genetically fused. The monomer and protein are geneticallyfused if the whole construct is expressed from a single polynucleotidesequence. Genetic fusion of a pore to a polynucleotide binding proteinis discussed in International Application No. PCT/GB09/001679 (publishedas WO 2010/004265).

If the polynucleotide binding protein is attached via cysteine linkage,the one or more cysteines have preferably been introduced to the mutantby substitution. Such substitutions are typically made in loop regionswhich have low conservation amongst homologues indicating that mutationsor insertions may be tolerated. They are therefore suitable forattaching a polynucleotide binding protein. Such substitutions aretypically made in residues 1 to 43 and 127 to 297 of SEQ ID NO: 2, Thereactivity of cysteine residues rimy be enhanced by modification asdescribed above.

The polynucleotide binding protein may be attached directly to themutant monomer or via one or more linkers. The polynucleotide bindingprotein may be attached to the mutant monomer using the hybridizationlinkers described in International Application No. PCT/GB10/000132(published as WO 2010/086602). Alternatively, peptide linkers may beused. Peptide linkers are amino acid sequences. The length, flexibilityand hydrophilicity of the peptide linker are typically designed suchthat it does not to disturb the functions of the monomer and molecule.Preferred flexible peptide linkers are stretches of 2 to 20, such as 4,6, 8, 10 or 16, serine and/or glycine amino acids. More preferredflexible linkers include (SG)₁, (SG)₂, (SG)₃, (SG)₄, (SG)₅ and (SG)₈wherein S is serine and G is glycine. Preferred rigid linkers arestretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids.More preferred rigid linkers include (P)₁₂ wherein P is proline.

The mutant monomer may be chemically modified with a molecular adaptorand a polynucleotide binding protein.

Making Mutant Lysenin Monomers

The invention also provides a method of improving the ability of alysenin monomer comprising the sequence shown in SEQ ID NO: 2 tocharacterise a polynucleotide. The method comprises making one or moremodifications within the region of from about position 44 to aboutposition 126 of SEQ ID NO: 2 which alter the ability of the monomer tointeract with a polynucleotide and do not affect time ability of themonomer to form a pore. Any of the embodiments discussed above withreference to the mutant lysenin monomers and below with reference tocharacterising polynucleotides equally apply to this method of theinvention.

Constructs

The invention also provides a construct comprising two or morecovalently attached monomers derived from lysenin wherein at least oneof the monomers is a mutant lysenin monomer of the invention. Theconstruct of the invention retains its ability to form a pore. One ormore constructs of the invention may be used to form pores forcharacterising a target analyte. One or more constructs of the inventionmay be used to form pores for characterising a target polynucleotide,such as sequencing a target polynucleotides. The construct may comprise2, 3, 4, 5, 6, 7, 8, 9 or 10 or more monomers. The two or more monomersmay be the same or different.

At least monomer in the construct is a mutant monomer of the invention.The other monomers in the construct do not have to be mutant monomers ofthe invention. For instance, at least one monomer may comprise thesequence shown in SEQ ID NO: 2. At least one monomer in the constructmay be a paralogue or homologue of SEQ ID NO: 2. Suitable homologues areshown in SEQ ID NOs: 16 to 19.

Alternatively, at least one monomer may comprise a variant of SEQ ID NO:2 which is at least 50% homologous to SEQ ID NO: 2 over its entiresequence based on amino acid identity, but does not include any of thespecific mutations required by the mutant monomers of the invention.More preferably, the variant may be at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90% and more preferably at least 95%, 97% or 99% homologous based onamino acid identity to the amino acid sequence of SEQ IID NO: 2 over theentire sequence. The variant may be a fragment or any other variantdiscussed above. Constructs of the invention may also comprise a variantof SEQ ID NO: 16, 17, 18 or 19 which is at least 50% homologous or atleast any of the other level of homology mentioned above to SEQ ID NO:16, 17, 18 or 19 over its entire sequence based on amino acid identity.

All of the monomers in the construct may be a mutant monomer of theinvention. The mutant monomers may be the same or different. In a morepreferred embodiment, the construct comprises two monomers and at leastone of the monomers is a mutant monomer of the invention.

The monomers may be genetically fused. Monomers are genetically fused ifthe whole construct is expressed from a single polynucleotide sequence.The coding sequences of the monomers may be combined in any way to forma single polynucleotide sequence encoding the construct. Genetic fusionis discussed in International Application No. PCT/GB09/001679 (publishedas WO 2010/004265).

The monomers may be genetically fused in any configuration. The monomersmay be fused via their terminal amino acids. For instance, the aminoterminus of the one monomer may be fused to the carboxy terminus ofanother monomer.

The two or more monomers may be genetically fused directly together. Themonomers are preferably genetically fused using a linker. The linker maybe designed to constrain the mobility of the monomers. Preferred linkersare amino acid sequences (i.e. peptide linkers). Any of the peptidelinkers discussed above may be used.

In another preferred embodiment, the monomers are chemically fused.Monomers are chemically fused if they are chemically attached, forinstance via a chemical crosslinker. Any of the chemical crosslinkersdiscussed above may be used. The linker may be attached to one or morecysteine residues or non-natural amino acids, such as Faz, introducedinto a mutant monomer Alternatively, the linker may be attached to aterminus of one of the monomers in the construct. Monomers are typicallylinked via one or more of residues 1 to 43 and 127 to 297 of SEQ ID NO:2.

If a construct contains different monomers, crosslinkage of monomers tothemselves may be prevented by keeping the concentration of linker in avast excess of the monomers. Alternatively, a “lock and key” arrangementmay be used in which two linkers are used. Only one end of each linkermay react together to form a longer linker and the other ends of thelinker each react with a different monomers. Such linkers are describedin International Application No. PCT/GB10/000132 (published as WO2010/086602).

The invention also provides a method of producing a construct of theinvention. The method comprises covalently attaching at least one mutantlysenin monomer of the invention to one or more monomers derived fromlysenin. Any of the embodiments discussed above with reference to theconstruct of the invention equally apply to the methods of producing theconstructs.

Polynucleotides

The present invention also provides polynucleotide sequences whichencode a mutant monomer of the invention. The mutant monomer may be anyof those discussed above. The polynucleotide sequence preferablycomprises a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologousbased on nucleotide identity to the sequence of SEQ ID NO: 1 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95% nucleotide identity over a stretch of 300 or more, forexample 375, 450, 525 or 600 or more, contiguous nucleotides (“hardhomology”). Homology may be calculated as described above. Thepolynucleotide sequence may comprise a sequence that differs from SEQ IDNO: 1 on the basis of the degeneracy of the genetic code.

The present invention also provides polynucleotide sequences whichencode any of the genetically fused constructs of the invention. Thepolynucleotide preferably comprises two or more sequences as shown inSEQ ID NO: 1 or a variant thereof as described above.

Polynucleotide sequences may be derived and replicated using standardmethods in the art. Chromosomal DNA encoding wild-type Lysenin may beextracted from a pore producing organism, such as Eisema fetida. Thegene encoding the pore monomer may be amplified using PCR involvingspecific primers. The amplified sequence may then undergo site-directedmutagenesis. Suitable methods of site-directed mutagenesis are known inthe art and include, for example, combine chain reaction.Polynucleotides encoding a construct of the invention can be made usingwell-known techniques, such as those described in Sambrook, J. andRussell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The resulting polynucleotide sequence may then be incorporated into arecombinant replicable vector such as a cloning vector. The vector maybe used to replicate the polynucleotide in a compatible host cell. Thuspolynucleotide sequences may be made by introducing a polynucleotideinto a replicable vector, introducing the vector into a compatible hostcell, and growing the host cell under conditions which bring aboutreplication of the vector. The vector may be recovered from the hostcell. Suitable host cells for cloning of polynucleotides are known inthe an and described in more detail below.

The polynucleotide sequence may be cloned into suitable expressionvector. In an expression vector, the polynucleotide sequence istypically operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by the host cell.Such expression vectors can be used to express a pore subunit.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. Multiple copies of the same or different polynucleotidesequences may be introduced into the vector.

The expression vector may then be introduced into a suitable host cell.Thus, a mutant monomer or construct of the invention can be produced byinserting a polynucleotide sequence into an expression vector,introducing the vector into a compatible bacterial host cell, andgrowing the host cell under conditions which bring about expression ofthe polynucleotide sequence. The recombinantly-expressed monomer orconstruct may self-assemble into a pore in the host cell membrane.Alternatively, the recombinant pore produced in this manner may beremoved from the host cell and inserted into another membrane. Whenproducing pores comprising at least two different subunits, thedifferent subunits may be expressed separately in different host cellsas described above, removed from the host cells and assembled into apore in a separate membrane, such as a sheep erythrocyte membrane orliposomes containing sphingomyelin.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide sequence and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example a tetracycline resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. A T7, trc, lac,ara or λ_(L), promoter is typically used.

The host cell typically expresses the pore subunit at a high level. Hostcells transformed with a polynucleotide sequence will be chosen to becompatible with the expression vector used to transform the cell. Thehost cell is typically bacterial and preferably Escherichia coli. Anycell with a λ DE3 lysogen, for example C41 (DE3), BL21 (DE3), JM109(DE3), B834 (DE3), TUNER, Origami and Origami B, can express a vectorcomprising the T7 promoter. In addition to the conditions listed aboveany of the methods cited in Proc Natl Acad Sci USA. 2008 December 30;105(52):20647-52 may be used to express the lysenin proteins.

Pores

The invention also provides various pores. The pores of the inventionare ideal for characterising analytes. The pores of the invention areespecially ideal for characterising, such as sequencing, polynucleotidesbecause they can discriminate between different nucleotides with a highdegree of sensitivity. The pores can be used to characterise nucleicacids, such as DNA and RNA, including sequencing the nucleic acid andidentifying single base changes. The pores of the invention can evendistinguish between methylated and unmethylated nucleotides. The baseresolution of pores of the invention is surprisingly high. The poresshow almost complete separation of all four DNA nucleotides. The porescan be further used to discriminate between deoxycytidine monophosphate(dCMP) and methyl-dCMP based on the dwell time in the pore and thecurrent flowing through the pore.

The pores of the invention can also discriminate between differentnucleotides under a range of conditions. In particular, the pores willdiscriminate between nucleotides under conditions that are favourable tothe characterising, such as sequencing, of polynucleotides. The extentto which the pores of the invention can discriminate between differentnucleotides can be controlled by altering the applied potential, thesalt concentration, the buffer, the temperature and the presence ofadditives, such as urea, betaine and DTT. This allows the function ofthe pores to be fine-tuned, particularly when sequencing. This isdiscussed in more detail below. The pores of the invention may also beused to identify polynucleotide polymers from the interaction with oneor more monomers rather than on a nucleotide by nucleotide basis.

A pore of the invention may be isolated, substantially isolated,purified or substantially purified. A pore of the invention is isolatedor purified if it is completely free of any other components, such aslipids or other pores. A pore is substantially isolated if it is mixedwith carriers or diluents which will not interfere with its intendeduse. For instance, a pore is substantially isolated or substantiallypurified if it is present in a form that comprises less than 10%, lessthan 5%, less than 2% or less than 1% of other components, such aslipids or other pores. Alternatively, a pore of the invention may bepresent in a lipid bilayer.

A pore of the invention may be present as an individual or single pore.Alternatively, a pore of the invention may be present in a homologous orheterologous population or plurality of two or more pores.

Homo-Oligomeric Pores

The invention also provides a homo-oligomeric pore derived from lysenincomprising identical mutant monomers of the invention. The monomers areidentical in terms of their amino acid sequence. The homo-oligomericpore of the invention is ideal for characterising, such as sequencing,polynucleotides. The honro-oligomeric pore of the invention may have anyof the advantages discussed above. The advantages of specifichorno-oligomeric pores of the invention are indicated in the Examples.

The homo-oligomeric pore may contain any number of mutant monomers. Thepore typically comprises two or more mutant monomers. One or more of themutant monomers is preferably chemically modified as discussed above. Inother words, one or more of the monomers being chemically modified (andthe others not being chemically modified) does not prevent the pore frombeing homo-oligorneric as long as the amino acid sequence of each of themonomers is identical.

Methods for making lysenin pores are described in the Examples and inYamaji et al., J. Biol. Chem. 1998; 273(9): 5300-6.

Hetero-Oligomeric Pores

The invention also provides a hetero-oligomeric pore derived fromlysenin comprising at least one mutant monomer of the invention, whereinat least one of the monomers differs from the others. The monomerdiffers from the others in teens of its amino acid sequence. Thehetero-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. Hetero-oligomeric pores can be madeusing methods known in the art (e.g. Protein Sci. 2002 July;11(7):1813-24).

The hetero-oligomeric pore contains sufficient monomers to form thepore. The monomers may be of any type. The pore typically comprises twoor more monomers.

The pore may comprise at least one monomer comprising the sequence shownin SEQ ID NO: 2, a paralogue thereof, a homologue thereof or a variantthereof which does not have a mutation required by the mutant monomersof the invention. Suitable variants are any of those discussed abovewith reference to the constructs of the invention, including SEQ ID NOs:2, 16, 17, 18 and 19 and variants thereof. In this embodiment, theremaining monomers are preferably mutant monomers of the invention.

In a preferred embodiment, the pore comprises (a) one mutant monomer ofthe invention and (b) a sufficient number of identical monomers to formthe pore, wherein the mutant monomer in (a) is different from theidentical monomers in (b). The identical monomers in (b) preferablycomprise the sequence shown in SEQ ID NO: 2, a paralogue thereof, ahomologue thereof or a variant thereof which does not have a mutationrequired by the mutant monomers of the invention.

A hetero-oligomeric pore of the invention preferably comprises only onemutant lysenin monomer of the invention.

In another preferred embodiment, all of the monomers in thehetero-oligomeric pore are mutant monomers of the invention and at leastone of them differs from the others.

In all the embodiments discussed above, one or more of the mutantmonomers is preferably chemically modified as discussed above. Thepresence of a chemical modification on one monomer does not result inthe pore being hetero-oligomeric. The amino acid sequence of at leastone monomer must differ from the sequence(s) of the other monomers.Methods for making pores are discussed in more detail below.

Construct-Containing Pores

The invention also provides a pore comprising at least one construct ofthe invention. A construct of the invention comprises two or morecovalently attached monomers derived from lysenin, wherein at least oneof the monomers is a mutant lysenin monomer of the invention. In otherwords, a construct must contain more than one monomer. At least two ofthe monomers in the pore are in the form of a construct of theinvention. The monomers may be of any type.

A pore typically contains (a) one construct comprising two monomers and(b) a sufficient number of monomers to form the pore. The construct maybe any of those discussed above. The monomers may be any of thosediscussed above, including mutant monomers of the invention.

Another typical pore comprises more than one construct of the invention,such as two, three or four constructs of the invention. Such poresfurther comprise a sufficient number of monomers to form the pore. Themonomer may be any of those discussed above. A further pore of theinvention comprises only constructs comprising 2 monomers. A specificpore according to the invention comprises several constructs eachcomprising two monomers. The constructs may oligomerise into a pore witha structure such that only one monomer from each construct contributesto the pore. Typically, the other monomers of the construct (i.e. theones not forming the pore) will be on the outside of the pore.

Mutations can be introduced into the construct as described above. Themutations may be alternating, i.e. the mutations are different for eachmonomer within a two monomer construct and the constructs are assembledas a homo-oligomer resulting in alternating modifications. In otherwords, monomers comprising MutA and MutB are fused and assembled to forman A-B:A-B:A-B:A-B pore. Alternatively, the mutations may beneighbouring, i.e. identical mutations are introduced into two monomersin a construct and this is then oligomerised with different mutantmonomers. In other words, monomers comprising MutA are fused follow byoligomerisation with MutB-containing monomers to form A-A:B:B:B:B:B:B.

One or more of the monomers of the invention in a construct-containingpore may be chemically-modified as discussed above.

Producing Pores of the Invention

The invention also provides a method of producing a pore of theinvention. The method comprises allowing at least one mutant monomer ofthe invention or at least one construct of the invention to oligomerisewith a sufficient number of mutant lysenin monomers of the invention,constructs of the invention or monomers derived from lysenin to form apore. If the method concerns making a homo-oligomeric pore of theinvention, all of the monomers used in the method are mutant lyseninmonomers of the invention having the same amino acid sequence. If themethod concerns making a hetero-oligorneric pore of the invention, atleast one of the monomers is different from the others. Any of theembodiments discussed above with reference to the pores of the inventionequally apply to the rnethods of producing the pores.

A preferred way of making a pore of the invention is disclosed inExample 1.

Methods of Characterising Analytes

The invention provides a method of characterising a target analyte. Themethod comprises contacting the target analyte with a pore of theinvention such that the target analyte moves through the pore. One ormore characteristics of the target analyte are then measured as theanalyte moves with respect to the pore using standard methods known inthe art. One or more characteristics of the target analyte arepreferably measured as the analyte moves through the pore. Steps (a) and(b) are preferably carried out with a potential applied across the pore.As discussed in more detail below, the applied potential typicallyresults in the formation of a complex between the pore and apolynucleotide binding protein. The applied potential may be a voltagepotential. Alternatively, the applied potential may be a chemicalpotential. An example of this is using a salt gradient across anamphiphilic layer. A salt gradient is disclosed in Holden et al., J AmChem. Soc. 2007 July 11; 129(27):8650-5.

The method of the invention is for characterising a target analyte. Themethod is for characterising at least one analyte. The method mayconcern characterising two or more analytes. The method may comprisecharacterising any number of analytes, such as 2, 5, 10, 15, 20, 30, 40,50, 100 or more analytes.

The target analyte is preferably a metal ion, an inorganic salt, apolymer, an amino acid, a peptide, a polypeptide, a protein, anucleotide, an oligonucleotide, a polynucleotide, a dye, a bleach, apharmaceutical, diagnostic agent, a recreational drug, an explosive oran environmental pollutant. The method may concern characterising two ormore analytes of the same type, such as two or more proteins, two ormore nucleotides or two or more pharmaceuticals. Alternatively, themethod may concern characterising two or more analytes of differenttypes, such as one or more proteins, one or more nucleotides and one ormore pharmaceuticals.

The target analyte can be secreted from cells. Alternatively, the targetanalyte can be an analyte that is present inside cells such that theanalyte must be extracted from the cells before the invention can becarried out.

The analyte is preferably an amino acid, a peptide, a polypeptidesand/or a protein. The amino acid, peptide, polypeptide or protein can benaturally-occurring or non-naturally-occurring. The polypeptide orprotein can include within them synthetic or modified amino acids. Anumber of different types of modification to amino acids are known inthe art. Suitable amino acids and modifications thereof are above. Forthe purposes of the invention, it is to be understood that the targetanalyte can be modified by any method available in the art.

The protein can be an enzyme, an antibody, a hormone, a growth factor ora growth regulatory protein, such as a cytokine. The cytokine may beselected from interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5,IL-6, IL-10, L-12 and IL-13, interferons, preferably IL-γ, and othercytokines such as TNF-α. The protein may be a bacterial protein, afungal protein, a virus protein or a parasite-derived protein.

The target analyte is preferably a nucleotide, an oligonucleotide or apolynucleotide. A nucleotide typically contains a nucleobase, a sugarand at least one phosphate group. The nucleobase is typicallyheterocyclic. Nucleobases include, but are not limited to, purines andpyrimidines and more specifically adenine, guanine, thytnine, uracil andcytosine. The sugar is typically a pentose sugar. Nucleotide sugarsinclude, but are not limited to, ribose and deoxyribose. The nucleotideis typically a ribonucleotide or deoxyribonucleotide. The nucleotidetypically contains a monophosphate, diphosphate or triphosphate.Phosphates may be attached on the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(MP), thymidine triphosphate (TTP), Uricline monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), 5-methyleytidine rnonophosphate, 5-methylcytidine diphosphate,5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate,5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidinetriphosphate cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP),5-methyl-2′-deoxycytidine monophosphate, 5-methyl-2′-deoxycytidinediphosphate, 5-methyl-2′-deoxycytidine triphosphate,5-hydroxymethyl-2′-deoxycytidine monophosphate,5-hydroxymethyl-2′-deoxycytidine diphosphate and5-hydroxymethyl-2′-deoxycytidine triphosphate. The nucleotides arepreferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.The nucleotides may be abasic (i.e. lack a nucleobase). The nucleotidesmay contain additional modifications. In particular, suitable modifiednucleotides include, but are not limited to, 2′amino pyrimidines (suchas 2′-amino cytidine and 2′-amino uridine), 2′-hyrdroxyl purines (suchas, 2′-fluoro pyrimidines (such as 2′-fluorocytidine and 2′fluorouridine), hydroxyl pyrimidines (such as 5′-α-P-borano uridine),2′-O-methyl nucleotides (such as 2′-O-methyl adenosine, 2′-O-methylguanosine, 2′-O-methyl cytidine and 2′-O-methyl uridine), 4′-thiopyrimidines (such as 4′-thio uridine and 4′-thio cytidine) andnucleotides have modifications of the nucleobase (such as5-pentynyl-2′-deoxy uridine, 5-(3-aminopropyl)-uridine and1,6-diaminohexyl-N-5-carbamoylmethyl uridine).

Oligonucleotides are short nucleotide polymers which typically have 50or fewer nucleotides, such 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer or 5 or fewer nucleotides. The oligonucleotides may comprise anyof the nucleotides discussed above, including the abasic and modifiednucleotides. The method of the invention is preferably forcharacterising a target polynucleotide, A polynucleotide, such as anucleic acid, is a macromolecule comprising two or more nucleotides. Thepolynucleotide or nucleic acid may comprise any combination of anynucleotides. The nucleotides can be naturally occurring or artificial.One or more nucleotides in the target polynucleotide can be oxidized ormethylated. One or more nucleotides in the target polynucleotide may bedamaged. For instance, the polynucleotide may comprise a pyrimidinedimer. Such dimers are typically associated with damage by ultravioletlight and are the primary cause of skin melanomas. One or morenucleotides in the target polynucleotide may be modified, for instancewith a label or a tag. Suitable labels are described above. The targetpolynucleotide may comprise one or more spacers.

Nucleotides are defined above. Nucleotides present in the polynucleotidetypically include, but are not limited to, adenosine monophosphate(AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP),uridine monophosphate (UMP), cytidine monophosphate (CMP), cyclicadenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP),deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate(dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate(dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides arepreferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMPand dUMP.

A nucleotide may be abasic (i.e. lack a nucleobase).

The nucleotides in the polynucleotide may be attached to each other inany manner. The nucleotides are typically attached by their sugar andphosphate groups as in nucleic acids. The nucleotides may be connectedvia their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded. A singlestranded polynucleotide may have one or more primers hybridised theretoand hence comprise one or more short regions of double strandedpolynucleotide. The primers may be the same type of polynucleotide asthe target polynucleotide or may be a different type of polynucleotide.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The target polynucleotide can compriseone strand of RNA hybridized to one strand of DNA. The polynucleotidemay be any synthetic nucleic acid known in the art, such as peptidenucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid(TNA), locked nucleic acid (LNA) or other synthetic polymers withnucleotide side chains.

The whole or only part of the target polynucleotide may be characterisedusing this method. The target polynucleotide can be any length. Forexample, the polynucleotide can be at least 10, at least 50, at least100, at least 150, at least 200, at least 250, at least 300, at least400 or at least 500 nucleotide pairs in length. The polynucleotide canbe 1000 or more nucleotide pairs, 5000 or more nucleotide pairs inlength or 100000 or more nucleotide pairs in length.

The target analyte, such as a target polynucleotide, is present in anysuitable sample. The invention is typically carried out on a sample thatis known to contain or suspected to contain the target analyte, such asthe target polynucleotide. Alternatively, the invention may be carriedout on a sample to confirm the identity of one or more target analytes,such as one or more target polynucleotides, whose presence in the sampleis known or expected.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaean,prokaryotic or eukaryotic and typically belongs to one the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum. Typically, the sample is human in origin, but alternatively itmay be from another mammal animal such as from commercially farmedanimals such as horses, cattle, sheep or pigs or may alternatively bepets such as cats or dogs. Alternatively a sample of plant origin istypically obtained from a commercial crop, such as a cereal, legume,fruit or vegetable, for example wheat, barley, oats, canola, maize,soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans,lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of a non-biological sample includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being assayed, for example bycentrifugation or by passage through a membrane that filters outunwanted molecules or cells, such as red blood cells. The sample may bemeasured immediately upon being taken. The sample may also be typicallystored prior to assay, preferably below −70° C.

The pore is typically present in a membrane. Any membrane may be used inaccordance with the invention. Suitable membranes are well-known in theart. The membrane preferably comprises sphingomyelin. The membrane ispreferably an amphiphilic layer. An amphiphilic layer is a layer formedfrom amphiphilic molecules, such as phospholipids, which have both atleast one hydrophilic portion and at least one lipophilic or hydrophobicportion. The amphiphilic molecules may be synthetic or naturallyoccurring. Non-naturally occurring amphiphiies and arnphiphites whichform a monolayer are known in the art and include, for example,blockcopolymers (Gonzalez-Perez et al., Langmuir, 2009, 25,10447-10450). Block copolymers are polymeric materials in which two ormore monomer sub-units that are polymerized together to create a singlepolymer chain. Block copolymers typically have properties that arecontributed by each monomer sub-unit. However, a block copolymer mayhave unique properties that polymers fortned from the individualsub-units do not possess. Block copolymers can be engineered such thatone of the monomer sub-units is hydrophobic (i.e. lipophilic), whilstthe other sub-unit(s) are hydrophilic whilst in aqueous media. In thiscase, the block copolymer may possess amphiphilic properties and mayform a structure that mimics a biological membrane. The block copolymermay be a diblock (consisting of two monomer sub-units), but may also beconstructed from more than two monomer sub-units to form more complexarrangements that behave as amphipiles. The copolymer may be a triblock,tetrablock or pentablock copolymer.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically a planar lipid bilayer or a supported bilayer.

The amphiphilic layer is typically a lipid bilayer. Lipid bilayers aremodels of cell membranes and serve as excellent platforms for a range ofexperimental studies. For example, lipid bilayers can be used for invitro investigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Example. Lipid bilayers are commonly formedby the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972,69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interlace.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734). In another preferred embodiment, the membrane is a solidstate layer. A solid-state layer is not of biological origin. In otherwords, a solid state layer is not derived from or isolated from abiological environment such as an organism or cell, or a syntheticallymanufactured version of a biologically available structure. Solid statelayers can be formed from both organic and inorganic materialsincluding, but not limited to, microelectronic materials, insulatingmaterials such as Si₃N₄, Al₂O₃, and SiO, organic and inorganic polymerssuch as polyimide, plastics such as Teflon® or elastomers such astwo-component addition-cure silicone rubber, and glasses. The solidstate layer may be formed from monatomic layers, such as graphene, orlayers that are only a few atoms thick. Suitable graphene layers aredisclosed in International Application No. PCT/US2008/010637 (publishedas WO 2009/035647).

The method is typically carried out using (i) an artificial amphiphiliclayer comprising a pore, (ii) an isolated, naturally-occurring lipidbilayer comprising a pore, or (iii) a cell having a pore insertedtherein. The method is typically carried out using an artificialamphiphilic layer, such as an artificial lipid bilayer. The layer maycomprise other transmembrane and/or intramembrane proteins as well asother molecules in addition to the pore. Suitable apparatus andconditions are discussed below. The method of the invention is typicallycarried out in vitro. The analyte, such as a target polynucleotide, maybe coupled to the membrane. This may be done using any known method. Ifthe membrane is an amphiphilic layer, such as a lipid bilayer (asdiscussed in detail above), the analyte, such as a targetpolynucleotide, is preferably coupled to the membrane via a polypeptidepresent in the membrane or a hydrophobic anchor present in the membrane.The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbonnanotube or amino acid.

The analyte, such as a target polynucleotide, may be coupled directly tothe membrane. The analyte, such as a target polynucleotide, ispreferably coupled to the membrane via a linker. Preferred linkersinclude, but are not limited to, polymers, such as polynucleotides,polyethylene glycols (PEGs) and polypeptides. If a polynucleotide iscoupled directly to the membrane, then some data will be lost as thecharacterising run cannot continue to the end of the polynucleotide dueto the distance between the membrane and the interior of the pore. If alinker is used, then the polynucleotide can be processed to completion.If a linker is used, the linker may be attached to the polynucleotide atany position. The linker is preferably attached to the polynucleotide atthe tail polymer.

The coupling may be stable or transient. For certain applications, thetransient nature of the coupling is preferred. If a stable couplingmolecule were attached directly to either the 5′ or 3′ end of apolynucleotide, then some data will be lost as the characterising runcannot continue to the end of the polynucleotide due to the distancebetween the bilayer and the interior of the pore. If the coupling istransient, then when the coupled end randomly becomes free of thebilayer, then the polynucleotide can be processed to completion.Chemical groups that form stable or transient links with the membraneare discussed in more detail below. The analyte, such as a targetpolynucleotide, may be transiently coupled to an amphiphilic layer, suchas a lipid bilayer using cholesterol or a fatty acyl chain. Any fattyacyl chain having a length of from 6 to 30 carbon atoms, such ashexadecanoic acid, may be used.

In preferred embodiments, the analyte, such as a target polynucleotide,is coupled to an amphiphilic layer. Coupling of analytes, such as atarget polynucleotide, to synthetic lipid bilayers has been carried outpreviously with various different tethering strategies. These aresummarised in Table 3 below.

TABLE 3 Attachment group Type of coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol- based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Lipid Stable van Lengerich,B., R. J. Rawle, et al. “Covalent attachment of lipid vesicles to afluid-supported bilayer allows observation of DNA-mediated vesicleinteractions.” Langmuir 26(11): 8666-72

Polynucleotides may be functionalized using a modified phosphorarniditein the synthesis reaction, which is easily compatible for the additionof reactive groups, such as thiol, cholesterol, lipid and biotin groups.These different attachment chemistries give a suite of attachmentoptions for polynucleotides. Each different modification group tethersthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the bilayer. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides can also be achieved by a number of othermeans provided that a reactive group can be added to the polynucleotide.The addition of reactive groups to either end of DNA has been reportedpreviously. A thiol group can be added to the 5′ of ssDNA usingpolynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). “Afacile method for attaching nitroxide spin labels at the 5′ terminus ofnucleic acids.” Nucleic Acids Res 35(10) e77). A more diverse selectionof chemical groups, such as biotin, thiols and fluorophores, can beadded using terminal transferase to incorporate modifiedoligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tchen, et al. (1988).“Nonradioactive labeling of synthetic oligonucleotide probes withterminal deoxynucleotidyl transferase,” Anal Biochem 169(2): 376-82).

Alternatively, the reactive group could be considered to be the additionof a short piece of DNA complementary to one already coupled to thebilayer, so that attachment can be achieved via hybridisation. Ligationof short pieces of ssDNA. have been reported using T4 RNA ligase I(Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Nati Acad Sci USA 89(20): 9823-5).Alternatively either ssDNA or dsDNA could be ligated to native dsDNA andthen the two strands separated by thermal or chemical denaturation. Tonative dsDNA, it is possible to add either a piece of ssDNA to one orboth of the ends of the duplex, or dsDNA to one or both ends. Then, whenthe duplex is melted, each single strand will have either a 5′ or 3′modification if ssDNA was used for ligation or a modification at the 5′end, the 3′ end or both if dsDNA was used for ligation. If thepolynucleotide is a synthetic strand, the coupling chemistry can beincorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesized using a primer areactive group attached to it.

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. By using an antisense primerthat has a reactive group, such as a cholesterol, thiol, biotin orlipid, each copy of the target DNA amplified will contain a reactivegroup for coupling.

The pore used in the method of the invention is a pore of the invention(i.e. a pore comprising at least one mutant monomer of the invention orat least one construct of the invention). The pore may be chemicallymodified in any of the ways discussed above. The pore is preferablymodified with a covalent adaptor that is capable of interacting with thetarget analyte as discussed above.

The method is preferably for characterising a target polynucleotide andstep (a) comprises contacting the target polynucleotide with the poreand a polynucleotide binding protein and the protein controls themovement of the target polynucleotide through the pore. Thepolynucleotide binding protein may be any protein that is capable ofbinding to the polynucleotide and controlling its movement through thepore. It is straightforward in the art to determine whether or not aprotein binds to a polynucleotide. The protein typically interacts withand modifies at least one property of the polynucleotide. The proteinmay modify the polynucleotide by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The moiety may modify the polynucleotide by orienting itor moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably a polynucleotidehandling enzyme. A polynucleotide handling enzyme is a polypeptide thatis capable of interacting with and modifying at least one property of apolynucleotide. The enzyme may modify the polynucleotide by cleaving itto form individual nucleotides or shorter chains of nucleotides, such asdi- or trinucleotides. The enzyme may modify the polynucleotide byorienting it or moving it to a specific position. The polynucleotidehandling enzyme does not need to display enzymatic activity as long asit is capable of binding the target sequence and controlling itsmovement through the pore. For instance, the enzyme may be modified toremove its enzymatic activity or may be used under conditions whichprevent it from acting as an enzyme. Such conditions are discussed inmore detail below.

The polynucleotide handling enzyme is preferably derived from anueleolytic enzyme. The polynucleotide handling enzyme used in theconstruct of the enzyme is more preferably derived from a member of anyof the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzymemay be any of those disclosed in International Application No.PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases andtopoisomerases, such as gyrases. Suitable enzymes include, but are notlimited to, exonuclease I from E. coli (SEQ ID NO: 6), exonuclease IIIenzyme from E. coli (SEQ ID NO: 8), Red from T. thermophilus (SEQ ID NO:10) and bacteriophage lambda exonuclease (SEQ ID NO: 12) and variantsthereof.

Three subunits comprising the sequence shown in SEQ ID NO: 10 or avariant thereof interact to form a trimer exonuclease. The enzyme may bePhi29 DNA polymerase (SEQ ID NO: 4) or a variant thereof. The enzyme maybe a helicase or derived from a helicase. Typical helicases are Hel308,RecD or XPD, for example Hel308 Mbu (SEQ ID NO: 15) or a variantthereof.

A variant of SEQ ID NOs: 4, 6, 8, 10, 12 or 15 is an enzyme that has anamino acid sequence which varies from that of SEQ ID NO: 4, 6, 8, 10, 12or 15 and which retains polynucleotide binding ability. The variant mayinclude modifications that facilitate binding of the polynucleotideand/or facilitate its activity at high salt concentrations and/or roomtemperature.

Over the entire length of the amino acid sequence of SEQ ID NO: 4, 6, 8,10, 12 or 15, a variant will preferably be at least 50% homologous tothat sequence based on amino acid identity. More preferably, the variantpolypeptide may be at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12 or 15over the entire sequence. There may be at least 80%, for example atleast 85%, 90% or 95%, amino acid identity over a stretch of 200 ormore, for example 230, 250, 270 or 280 or more, contiguous amino acids(“hard homology”). Homology is determined as described above. Thevariant may differ from the wild-type sequence in any of the waysdiscussed above with reference to SEQ ID NO: 2. The enzyme may becovalently attached to the pore as discussed above.

There are two main strategies for sequencing polynucleotides usingnanopores, namely strand sequencing and exonuclease sequencing. Themethod of the invention may concern either strand sequencing orexonuclease sequencing.

In strand sequencing, the DNA is translocated through the nanoporeeither with or against an applied potential. Exonucleases that actprogressively or processively on double stranded DNA can be used on thecis side of the pore to feed the remaining single strand through underan applied potential or the trans side under a reverse potential.Likewise, a helicase that unwinds the double stranded DNA can also beused in a similar manner. A polymerase may also be used. There are alsopossibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

In one embodiment, the method of characterising a target polynucleotideinvolves contacting the target sequence with a pore and a helicaseenzyme. Any helicase may be used in the method. Helicases may work intwo modes with respect to the pore. First, the method is preferablycarried out using a helicase such that it controls movement of thetarget sequence through the pore with the field resulting from theapplied voltage. In this mode the 5′ end of the DNA is first captured inthe pore, and the enzyme controls movement of the DNA into the pore suchthat the target sequence is passed through the pore with the field untilit finally translocates through to the trans side of the bilayer.Alternatively, the method is preferably carried out such that a helicaseenzyme controls movement of the target sequence through the pore againstthe field resulting from the applied voltage. In this mode the 3′ end ofthe DNA is first captured in the pore, and the enzyme controls movementof the DNA through the pore such that the target sequence is pulled outof the pore against the applied field until finally ejected back to thecis side of the bilayer.

In exonuclease sequencing, an exonuclease releases individualnucleotides from one end of the target polynucleotide and theseindividual nucleotides are identified as discussed below. In anotherembodiment, the method of characterising a target polynucleotideinvolves contacting the target sequence with a pore and an exonucleaseenzyme. Any of the exonuclease enzymes discussed above may be used inthe method. The enzyme may be covalently attached to the pore asdiscussed above.

Exonucleases are enzymes that typically latch e one end of apolynucleotide and digest the sequence one nucleotide at a time fromthat end. The exonuclease cart digest the polynucleotide in the 5′ to 3′direction or 3′ to 5′ direction. The end of the polynucleotide to whichthe exonuclease binds is typically determined through the choice ofenzyme used and/or using methods known in the art. Hydroxyl groups orcap structures at either end of the polynucleotide may typically be usedto prevent or facilitate the binding of the exonuclease to a particularend of the polynucleotide.

The method involves contacting the polynucleotide with the exonucleaseso that the nucleotides are digested from the end of the polynucleotideat a rate that allows characterisation or identification of a proportionof nucleotides as discussed above. Methods for doing this are well knownin the art. For example, Edman degradation is used to successivelydigest single amino acids from the end of polypeptide such that they maybe identified using High Performance Liquid Chromatography (HPLC). Ahomologous method may be used in the present invention.

The rate at which the exonuclease functions is typically slower than theoptimal rate of a wild-type exonuclease. A suitable rate of activity ofthe exonuclease in the method of the invention involves digestion offrom 0.5 to 1000 nucleotides per second, from 0.6 to 500 nucleotides persecond, 0.7 to 200 nucleotides per second, from 0.8 to 100 nucleotidesper second, from 0.9 to 50 nucleotides per second or 1 to 20 or 10nucleotides per second. The rate is preferably 1, 10, 100, 500 or 1000nucleotides per second. A suitable rate of exonuclease activity can beachieved in various ways. For example, variant exonucleases with areduced optimal rate of activity may be used in accordance with theinvention.

The method of the invention involves measuring one or morecharacteristics of the target analyte, such as a target polynucleotide.The method may involve measuring two, three, four or five or morecharacteristics of the target analyte, such as a target polynucleotide.For target polynucleotides, the one or more characteristics arepreferably selected from (i) the length of the target polynucleotide,(ii) the identity of the target polynucleotide, (iii) the sequence ofthe target polynucleotide, (iv) the secondary structure of the targetpolynucleotide and (v) whether or not the target polynucleotide ismodified. Any combination of (i) to (v) may be measured in accordancewith the invention.

For (i), the length of the polynucleotide may be measured using thenumber of interactions between the target polynucleotide and the pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the targetpolynucleotide or without measurement of the sequence of the targetpolynucleotide. The former is straightforward; the polynucleotide issequenced and thereby identified. The latter may be done in severalways. For instance, the presence of a particular motif in thepolynucleotide may be measured (without measuring the remaining sequenceof the polynucleotide). Alternatively, the measurement of a particularelectrical and/or optical signal in the method may identify the targetpolynucleotide as coming from a particular source.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not the targetpolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcyotsine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

The invention also provides a method of estimating the sequence of atarget polynucleotide. The invention further provides a method ofsequencing a target polynucleotide.

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements.Possible electrical measurements include: current measurements,impedance measurements, tunnelling measurements (Ivanov A P et al., NanoLett. 2011 Jan. 12; 11W:279-85), and FET measurements (InternationalApplication WO 2005/124888). Optical measurements may be combined 10with electrical measurements (Soni G V et al., Rev Sci Instrum. 2010January; 81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc, 2010;132(50):17961-72, and International Application WO-2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International ApplicationWO-2009/077734 and International Application WO-2011/067559.

In a preferred embodiment, the method comprises:

(a) contacting the target polynucleotide with a pore of the inventionand a polynucleotide binding protein such that the target polynucleotidemoves through the pore and the binding protein controls the movement ofthe target polynucleotide through the pore; and

(b) measuring the current passing through the pore as the polynucleotidemoves with respect to the pore wherein the current is indicative of oneor more characteristics of the target polynucleotide and therebycharacterising the target polynucleotide.

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore is inserted into amembrane. The method may be carried out using any apparatus that issuitable for transmembrane pore sensing. For example, the apparatuscomprises a chamber comprising an aqueous solution and a barrier thatseparates the chamber into two sections. The barrier has an aperture inwhich the membrane containing the pore is formed.

The methods may be carried out using the apparatus described ininternational Application No. PCT/GB08/000562 (WO 2008/102120).

The methods may involve measuring the current passing through the poreas the analyte, such as a target polynucleotide, moves with respect tothe pore. Therefore the apparatus may also comprise an electricalcircuit capable of applying a potential and measuring an electricalsignal across the membrane and pore. The methods may be carried outusing a patch clamp or a voltage clamp. The methods preferably involvethe use of a voltage clamp.

The methods of the invention may involve the measuring of a currentpassing through the pore as the analyte, such as a targetpolynucleotide, moves with respect to the pore. Suitable conditions formeasuring ionic currents through transmembrane protein pores are knownin the art and disclosed in the Example. The method is typically carriedout with a voltage applied across the membrane and pore. The voltageused is typically from +2 V to −2 V, typically −400 mV to +400 mV. Thevoltage used is preferably in a range having a lower limit selected from−400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0 mV andan upper limit independently selected from +10 mV, +20 mV, +50 mV, +100mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is morepreferably in the range 100 mV to 240 mV and most preferably in therange of 120 mV to 220 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The methods are typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl) or caesium chloride (CsCl) is typicallyused. KCl is preferred. The salt concentration may be at saturation. Thesalt concentration may be 3M or lower and is typically from 0.1 to 2.5M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from150 mM to 1 M. The method is preferably carried out using a saltconcentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M,at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrationsprovide a high signal to noise ratio and allow for currents indicativeof the presence of a nucleotide to be identified against the backgroundof normal current fluctuations.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is HEPES. Another suitable bufferis Tris-HCl buffer. The methods are typically carried out at a pH offrom 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8,from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used ispreferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to95° C. from 16° C. to 90° C., from 17 to 85° C., from 18° C. to 80° C.,19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

The method is typically carried out in the presence of free nucleotidesor free nucleotide analogues and an enzyme cofactor that facilitate theaction of the polynucleotide binding protein, such as a helicase or anexonuclease. The free nucleotides may be one or more of any of theindividual nucleotides discussed above. The free nucleotides include,but are not limited to, adenosine monophosphate (AMP), adenosinediphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate(GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP),thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidinetriphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate(UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP),cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclicadenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP),deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP),deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP),deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP),deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP),deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP),deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dLITP),deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) anddeoxycytidine triphosphate (dCTP). The free nucleotides are preferablyselected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. Thefree nucleotides are preferably adenosine triphosphate (ATP). The enzymecofactor is a factor that allows the helicase to function. The enzymecofactor is preferably a divalent metal cation. The divalent metalcation is preferably Mg²⁺, Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor ismost preferably Mg²⁺.

The target polynucleotide may be contacted with the pore and thepolynucleotide binding protein in any order. In is preferred that, whenthe target polynucleotide is contacted with the protein and the pore,the target polynucleotide firstly forms a complex with the protein. Whenthe voltage is applied across the pore, the targetpolynucleotide/protein complex then forms a complex with the pore andcontrols the movement of the polynucleotide through the pore.

Methods of Identifying an Individual Nucleotide

The present invention also provides a method of characterising anindividual nucleotide. In other words, the target analyte is aindividual nucleotide. The method comprises contacting the nucleotidewith a pore of the invention such that the nucleotide interacts with thepore and measuring the current passing through the pore during theinteraction and thereby characterising the nucleotide. The inventiontherefore involves nanopore sensing of an individual nucleotide. Theinvention also provides a method of identifying an individual nucleotidecomprising measuring the current passing through the pore during theinteraction and thereby determining the identity of the nucleotide. Anyof the pores of the invention discussed above may be used. The pore ispreferably chemically modified with a molecular adaptor as discussedabove.

The nucleotide is present if the current flows through the pore in amanner specific for the nucleotide (i.e. if a distinctive currentassociated with the nucleotide is detected flowing through the pore).The nucleotide is absent if the current does not flow through the porein a manner specific for the nucleotide.

The invention can be used to differentiate nucleotides of similarstructure on the basis of the different effects they have on the currentpassing through a pore. Individual nucleotides can be identified at thesingle molecule level from their current amplitude when they interactwith the pore. The invention can also be used to determine whether ornot a particular nucleotide is present in a sample. The invention canalso be used to measure the concentration of a particular nucleotide ina sample.

The pore is typically present in a membrane. The methods may be carriedout using any suitable membrane/pore system described above.

An individual nucleotide is a single nucleotide. An individualnucleotide is one which is not bound to another nucleotide orpolynucleotide by a nucleotide bond. A nucleotide bond involves one ofthe phosphate groups of a nucleotide being bound to the sugar group ofanother nucleotide. An individual nucleotide is typically one which isnot bound by a nucleotide bond to another polynucleotide of at least 5,at least 10, at least 20, at least 50, at least 100, at least 200, atleast 500, at least 1000 or at least 5000 nucleotides. For example, theindividual nucleotide has been digested from a target polynucleotidesequence, such as a DNA or RNA strand. The methods of the invention maybe used to identify any nucleotide. The nucleotide can be any of thosediscussed above.

The nucleotide may be derived from the digestion of a nucleic acidsequence such as ribonucleic acid (RNA) or deoxyribonucleic acid.Nucleic acid sequences can be digested using any method known in theart. Suitable methods include, but are not limited to, those usingenzymes or catalysts. Catalytic digestion of nucleic acids is disclosedin Deck et al., Inorg. Chem., 2002; 41: 669-677.

Individual nucleotides from a single polynucleotide may be contactedwith the pore in a sequential manner in order to sequence the whole orpart of the polynucleotide. Sequencing polynucleotides is discussed inmore detail above.

The nucleotide may be contacted with the pore on either side of themembrane. The nucleotide may be introduced to the pore on either side ofthe membrane. The nucleotide may be contacted with the side of themembrane that allows the nucleotide to pass through the pore to theother side of the membrane. For example, the nucleotide is contactedwith an end of the pore, which in its native environment allows theentry of ions or small molecules, such as nucleotides, into the barrelor channel of the pore such that the nucleotide may pass through thepore. In such cases, the nucleotide interacts with the pore and/oradaptor as it passes across the membrane through the barrel or channelof the pore. Alternatively, the nucleotide may be contacted with theside of the membrane that allows the nucleotide to interact with thepore via or in conjunction with the adaptor, dissociate from the poreand remain on the same side of the membrane. The present inventionprovides pores in which the position of the adaptor is fixed. As aresult, the nucleotide is preferably contacted with the end of the porewhich allows the adaptor to interact with the nucleotide.

The nucleotide may interact a the pore in any manner and at any site. Asdiscussed above, the nucleotide preferably reversibly binds to the porevia or in conjunction with the adaptor. The nucleotide most preferablyreversibly binds to the pore via or in conjunction with the adaptor asit passes through the pore across the membrane. The nucleotide can alsoreversibly bind to the barrel or channel of the pore via or inconjunction with the adaptor as it passes through the pore across themembrane.

During the interaction between the nucleotide and the pore, thenucleotide affects the current flowing through the pore in a mannerspecific for that nucleotide. For example, a particular nucleotide willreduce the current flowing through the pore for a particular mean timeperiod and to a particular extent in other words, the current flowingthrough the pore is distinctive for a particular nucleotide. Controlexperiments may be carried out to determine the effect a particularnucleotide has on the current flowing through the pore. Results fromcarrying out the method of the invention on a test sample can then becompared with those derived from such a control experiment in order toidentify a particular nucleotide in the sample or determine whether aparticular nucleotide is present in the sample. The frequency at whichthe current flowing through the pore is affected in a manner indicativeof a particular nucleotide can be used to determine the concentration ofthat nucleotide in the sample. The ratio of different nucleotides withina sample can also be calculated. For instance, the ratio of dCMP tomethyl-dCMP can be calculated.

The method may involve the use of the any apparatus, sample or conditiondiscussed above.

Methods of Forming Sensors

The invention also provides a method of forming a sensor forcharacterising a target polynucleotide. The method comprises forming acomplex between a pore of the invention and a polynucleotide bindingprotein, such as a helicase or an exonuclease. The complex may be formedby contacting the pore and the protein in the presence of the targetpolynucleotide and then applying a potential across the pore. Theapplied potential may be a chemical potential or a voltage potential asdescribed above. Alternatively, the complex may be formed by covalentlyattaching the pore to the protein. Methods for covalent attachment areknown in the art and disclosed, for example, in InternationalApplication Nos. PCT/GB09/001679 (published as WO 2010/004265) andPCT/GB10/000133 (published as WO 2010/086603). The complex is a sensorfor characterising the target polynucleotide. The method preferablycomprises forming a complex between a pore of the invention and ahelicase. Any of the embodiments discussed above equally apply to thismethod.

The invention also provides a sensor for characterising a targetpolynucleotide. The sensor comprises a complex between a pore of theinvention and a polynucleotide binding protein. Any of the embodimentsdiscussed above equally apply to the sensor of the invention.

Kits

The present invention also provides a kit for characterising, such assequencing, a target polynucleotide. The kit comprises (a) a pore of theinvention and (b) a polynucleotide binding protein, such as a helicaseor an exonuclease. Any of the embodiments discussed above equallyapplicable to the kits of the invention.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotidesequences, a membrane as defined above or voltage or patch clampapparatus. Reagents may be present in the kit in a dry state such that afluid sample resuspends the reagents. The kit may also, optionally,comprise instructions to enable the kit to be used in the method of theinvention or details regarding which patients the method may be usedfor. The kit may, optionally, comprise nucleotides.

Apparatus

The invention also provides an apparatus for characterising, such assequencing, target polynucleotides in a sample. The apparatus maycomprise (a) a plurality of pores of the invention and (b) a pluralityof polynucleotide binding, proteins, such as helicases or exonucleases.The apparatus may be any conventional apparatus for analyte analysis,such as an array or a chip.

The apparatus preferably comprises:

a sensor device that is capable of supporting the plurality of pores andbeing operable to perform polynucleotide characterising or sequencingusing the pores and proteins;

-   -   at least one reservoir for holding material for performing the        characterising or sequencing;    -   a fluidics system configured to controllably supply material        from the at least one reservoir to the sensor device; and    -   a plurality of containers for receiving respective samples, the        fluidics system being configured to supply the samples        selectively from the containers to the sensor device.

The apparatus may be any of those described in International ApplicationNo. PCT/GB10/000789 (published as WO 2010/122293), InternationalApplication No. PCT/GB10/002206 (not yet published) or InternationalApplication No. PCT/US99/25679 (published as WO 00/28312).

The following Examples illustrate invention:

Example 1 Pore Production

DNA Synthesis

The polypeptide for lysenin was synthesised at GenScript USA Inc. andcloned into a p17 vector using NdeI and HindIII restriction sites. Codonfor Met (ATG) was placed at the beginning of the DNA for expressionpurposes and two stop codons (TAA TGA) were placed at the end of the DNAto terminate translation.

Protein Expression and Oligomerisation

Protein was generated by coupled in vitro transcription and translation(IVTT) using an E. coli T7-S30 extract system for circular DNA. Proteinwas expressed in the presence of sphingomyelin (SM) containing lipidvesicles to facilitate oligomerisation upon expression of monomersunits. To prepare SM vesicles, 0.5 mL of 25 mg/mL stock solution of SM(Avanti Polar Lipids, Cat No. 860062C) in chloroform was left at 37° C.to evaporate off chloroform. Once chloroform has evaporated, 5 mL of TEbuffer (10 mM Tris, 1 mM EDTA, pH 8.0) was added to the vial tosolubilise lipids. Mixture was then vortexed for about 1 minute andflash frozen with nitrogen. Lipid mixture was then thawed at 37° C.,vortexed and flash frozen again. This was repeated 5-6 times. Togenerate 100 uL of IVTT protein in the presence of lipid vesicles, 25 μLof prepared SM lipid vesicles were pelleted by spinning at 20,000 g for10 minutes. Once the supernatant was removed, components of the IVTT kit(Invitrogen Expressway Maxi Expression Module, Cat No. 45-4001),Methionine L-[35S] (Perkin Elmer, product number NEG009A005MC, specificactivity: >1000Ci (37.0 TBc)/mMole) and DNA template were added to thepellet according to manufacturer's insinictions. Briefly, 20 uL of E.coli slyD-extract, 20 uL of 2.5× IVPS reaction buffer without aminoacids, 1.25 uL of 50 mM amino acids lacking methionine, 0.5 uL of 75 mMmethionine, 0.5 uL of Methionine L-[35S], 1.0 uL of T7 enzyme mix, 2.5uL of DNA template at 400 ng/uL (1 ug) and 4.25 uL of RNase free waterwere added to the membrane pellet and incubated at 37° C. for 30minutes. 50 uL of feed buffer containing 25 uL of 2× IVPS feed buffer,1.25 uL of 50 mM amino acids lacking methionine, 0.5 uL of 75 mMmethionine, 0.5 uL of Methionine L-[35S] and 22.75 uL of RNase freewater were then added to the mixture and incubated at 37° C. for anadditional 90 minutes. Sample was then spun at 20,000 g for 10 minutesand the supernatant removed. 100 uL of Laemmli loading buffer (1×)containing 3× SDS was added to the supernatant. The sample thensubjected to SDS-PAGE electrophoresis on a 7.5% gel.

Protein Purification

The gel was dried at 50° C. for 3 hours onto paper (Whatman 3MM Chr)under a vacuum, and exposed to an X-ray film overnight (about 18 hours).Using the autoradiograph as a template, protein oligomer band was cutfrom the dried gel. After rehydration in 150 uL TE buffer, the paper wasremoved. The gel was then crushed using a disposable pestle, and theslurry was filtered through a Costar spin-X centrifuge tube filters(0.22 μm pore CA membrane, product number 8160) by centrifugation at25,000 g for 10 min. The protein solution (filtrate) was then taken touse in planar lipid bilayer experiments.

Using an analogous procedure to that described above in Example 1, thefollowing lysenin mutants were made and purified:—Lysenin-(E84Q/E85K/E92Q/E97S/D126G/E135S) (SEQ ID NO: 2 with mutationsE84Q/E85K/E92Q/E97S/D126G/E135S), Lysenin-(E85K/E92Q/E94S/E97S/D126G)(SEQ ID NO: 2 with mutations E85K/E92Q/E94S/E97S/D126G),Lysenin-(E76S/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsE76S/E85K/E92Q/E97S/D126G), Lysenin-(E71S/E85K/E92Q/E97S/D126G) (SEQ ID)NO: 2 with mutations E71S/E85K/E92Q/E97S/D126G),Lysenin-(D68S/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsD68S/E85K/E92Q/E97S/D126G), Lysenin-(E85K/E92Q/E97S/D126G) (SEQ ID NO: 2with mutations E85K/E92Q/E97S/D126G),Lysenin-(E84Q/E85K1E92Q/E97S/H103S/D126G) (SEQ ID NO: 2 with mutationsE84Q/E85K/E92Q/E97S/H103S/D126G),Lysenin-(E84Q/E85K/M90S/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsE84Q/E85K/M90S/E92Q/E97S/D126G),Lysenin-(E84Q/Q87S/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsE84Q/Q87S/E85K/E92Q/E97S/D126G), Lysenin-(E84Q/E85S/E92Q/E97S/D126G)(SEQ ID NO: 2 with mutations ES84Q/E85S/E92Q/E97S/D126G),Lysenin-(E84S/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsE84S/E85K/E92Q/E97S/D126G), Lysenin-(H81S/E84Q/E85K/E92Q/E97S/D126G)(SEQ ID NO: 2 with mutations H81S/E84Q/E85K/E92Q/E97S/D126G),Lysenin(Y79S/E84Q/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsY79S/E84Q/E85K/E92Q/E97S/D126G),Lysenin-(F70S/E84Q/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsF70S/E84Q/E85K/E92Q/E97S/D126(1),Lysenin-(H58S/E84Q/E85K/E92Q/E97S/D126G) (SEQ ID NO: 2 with mutationsH58S/E84Q/E85K/E92Q/E97S/D126G), Lysenin-(E92Q/E97S) (SEQ ID NO: 2 withmutations E92Q/E97S), Lysenin-(E84Q/E85K/E92Q/E97S) (SEQ ID NO: 2 withmutations E84Q/E85K/E92Q/E97S), Lysenin-(E84Q/E85K/D126G) (SEQ ID NO. 2with mutations E84Q/E85K/D126G), Lysenin-(E84Q/E85K/D126G/E167A) (SEQ IDNO: 2 with mutations E84Q/E85K/D126G/E167A), Lysenin-(E92Q/E97S/D126G)(SEQ ID NO: 2 with mutations E92Q/E97S/D126G), Lysenin-(E84D/E85K/E92Q)(SEQ ID NO: 2 with mutations E84D/E85K/E92Q), Lysenin-(E84Q) (SEQ ID NO:2 with the mutation E84Q), Lysenin-(D126N) (SEQ ID NO. 2 with themutation D126N), Lysenin-(E92Q) (SEQ ID NO: 2 with the mutation E92Q).

Example 2

This Example illustrates that it was possible to observe pore insertionof wild-type lysenin (SEQ ID NO: 2) nanopores into1,2-diphytanoyl-glycero-3-phosphocholine lipid (DPhPC) bilayers. It wasnot possible to observe DNA capture events or any helicase controlledDNA movement under the experimental conditions tested. The generalmethod and substrate employed throughout this Example is shown in FIG. 1and described in the figure caption.

Materials and Methods

Primers were designed to amplify a ˜400 bp fragment of PhiX174. Each ofthe 5′-ends of these primers included a 50 nucleotide non-complementaryregion, either a homopolymeric stretch or repeating units of 10nucleotide homopolymeric sections. In addition, the 5′-end of theforward primer was “capped” to include four 2-O-Methyl-Uracil (mU)nucleotides and the 5′-end of the reverse primer was chemicallyphosphorylated. These primer modifications then allow for the controlleddigestion of predominantly only the antisense strand, using lambdaexonuclease. The mU capping protects the sense strand from nucleasedigestion whilst the PO4 at the 5′ of the antisense strand promotes it.Therefore after incubation with lambda exonuclease only the sense strandof the duplex remains intact, now as single stranded DNA (ssDNA). Thegenerated ssDNA was then PAGE purified as previously described.

The DNA substrate design used in all the experiments described here isshown in FIG. 2. The DNA substrate consists of a 400 base section ofssDNA. from PhiX, with a 50T 5′-leader. Annealed to this strand justafter the 50T leader is a pinker containing a 3′ cholesterol tag toenrich the DNA on the surface of the bilayer, and thus improve captureefficiency.

Electrical measurements were acquired from single wild-type lysenin (SEQID NO: 2) nanopores inserted in 1,2-diphytanoyl-glycero-3-phosphocholinelipid (DPhPC, Avanti Polar Lipids) bilayers. Bilayers were formed across˜100 μm diameter apertures in 20 μm thick PTFE films (in custom Delrinchambers) via the Montal-Mueller technique, separating two 1 mL bufferedsolutions. All experiments were carried out in the stated bufferedsolution. Single-channel currents were measured on Axopatch 200Bamplifiers (Molecular Devices) equipped with 1440 A digitizers. Platinumelectrodes were connected to the buffered solutions so that the ciscompartment (to which both nanopore and enzyme/DNA are added) isconnected to the ground of the Axopatch headstage, and the transcompartment is connected to the active electrode of the headstage.

After achieving a single wild-type lysenin (SEQ ID NO: 2) pore in thebilayer in buffer (625 mM KCl, 100 mM Hepes pH 8.0, 75 mM PotassiumFerrocyanide (II), 25 mM Potassium Ferricyanide (III), 10 mM MgCl₂), acontrol was run at +120 mV for 5 mins. DNA polynucleotide (SEQ ID NO: 13and 14) and Hel308 Mbu (SEQ ID NO: 15) were added to 50 μL of buffer(625 mM KCl, 100 mM Hepes pH 8.0, 75 mM Potassium Ferrocyanide (II), 25mM Potassium Ferricyanide (III), 10 mM MgCl₂) and pre-incubated for 5mins (DNA=6 nM, Enzyme (Hel308 Mbu)=2 μM). This pre-incubation mix wasadded to 950 μL of buffer (625 mM KCl, 100 mM Hepes pH 8.0, 75 mMPotassium Ferrocyanide (II), 25 mM Potassium Ferricyanide (III), 10 mMMgCl₂) in the cis compartment of the electrophysiology chamber to try toinitiate capture of the helicase-DNA complexes in the lysenin nanopore(to give final concentrations of DNA=0.3 nM, Enzyme (Hel308 Mbu)=100 mM(SEQ ID NO: 15)). Another control was run at +120 mV for 5 mins.helicase ATPase activity was initiated as required by the addition ofNTP (1 mM ATP) to the cis compartment. Experiments were carried out at aconstant potential of +120 mV.

Results and Discussion

It was possible to observe insertion of the WT lysenin (SEQ ID NO: 2)nanopores into the DPhPC bilayer (FIG. 3). A stable open pore current ofapproximately 280 pA was observed. However, upon the addition of thehelicase-DNA substrate mix to the cis compartment no DNA capture eventsor helicase controlled DNA movement was observed.

Example 3

This Example illustrates the use of a. Hel308 helicase (Hel308 MBu, SEQID NO: 15) to control the movement of intact DNA strands through amutant lysenin nanopore (Lys-E84D/E85K, SEQ ID NO: 2 with the mutationsE84D/E85K). The general method and substrate employed throughout thisExample is shown in FIG. 1 and described in the figure caption.

Electrical measurements were acquired as described in Example 2. Afterachieving a single lysenin-E84D/E851((SEQ ID NO: 2 with the mutationsE84D/E85K) pore in the bilayer under buffered conditions (625 mM KCl,100 mM Hepes pH 8.0, 75 mM, potassium ferrocyanide(II), 25 mM potassiumferricyanide(III)), MgCl₂ (10 mM) was added to the cis compartment and acontrol was run at +120 mV for 5 mins. DNA polynucleotide (SEQ ID NO: 13and 14) and Hel308 Mbu (SEQ ID NO: 15) were added to 50 tμL of buffer(625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II), 25mM potassium ferricyanide(III), 10 mM MgCl₂) and pre-incubated for 5mins (DNA=6 nM, Enzyme=2 μM). This pre-incubation mix was added to 950μL of buffer (625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassiumferrocyanide(II), 25 mM potassium ferricyanide(III), 10 mM MgCl₂) in thecis compartment of the electrophysiology chamber to initiate capture ofthe helicase-DNA complexes in the lysenin nanopore (to give finalconcentrations of DNA=0.3 nM, Enzyme=100 nM (SEQ ID NO: 15)). Anothercontrol was run at +120 mV for 10 mins. Helicase ATPase activity wasinitiated as required by the addition of NTP (1 mM ATP) to the ciscompartment. Experiments were carried out at a constant potential ofeither +120 or +180 mV.

Results and Discussion

The addition of Helicase-DNA substrate to Lysenin-E84D/E85K (SEQ ID NO:2 with the mutations E84D/E85K) as shown in FIG. 1 producescharacteristic current blocks as shown in FIG. 4 (at an appliedpotential of +180 mV). DNA without helicase bound interacts transientlywith the nanopore producing short-lived blocks in current (<<1 second).DNA with helicase bound and active (i.e. moving along the DNA strandunder ATPase action) produces long characteristic blocks levels withstepwise changes in current as shown in FIG. 4. Different DNA motifs inthe nanopore give rise to unique current block levels.

For a given substrate, we observe a characteristic pattern of currenttransitions that reflects the DNA sequence (examples in FIG. 4). Theevent range was observed to be approximately 25 pA (at an appliedpotential of +180 mV).

Example 4

This Example illustrates the use of a Hel308 helicase (Hel308 MBu, SEQID NO: 15) to control the movement of intact DNA strands through amutant lysenin nanopore (Lysenin-E92N/E94N/E97N/D121N/D126N, SEQ ID NO:2 with the mutations E92N/E94N/E97N/D121N/D126N). The general method andsubstrate employed throughout this Example is shown in FIG. 1 anddescribed in the figure caption.

Electrical measurements were acquired as described in Example 2. Afterachieving a single lysenin-E92N/E94N/E97N/D121N/D126N (SEQ ID NO: 2 withthe mutations E92N/E94N/E97N/D121N/D126N) nanopore in the bilayer underbuffered conditions (625 mM KCl, 100 μM Hepes pH 8.0, 75 mM potassiumferrocyanide(II), 25 mM potassium ferricyanide(III)), MgCl₂ (10 mM) wasadded to the cis compartment and a control was run at +120 mV for 5mins. DNA polynucleotide (SEQ ID NO: 13 and 14) and Hel308 Mbu (SEQ NO:15) were added to 50 μL of buffer (625 mM KCl, 100 mM Hepes pH 8.0, 75mM potassium ferrocyanide(II), 25 mM potassium ferricyanide(III), 10 MMMgCl₂) and pre-incubated for 5 mins (DNA=6 nM, Enzyme=2 μM). Thispre-incubation mix was added to 950 μL of buffer (625 mM KCl, 100 mMHepes pH 8.0, 75 mM potassium ferrocyanide(II), 25 mM potassiumferricyanide(III), 10 mM MgCl₂) in the cis compartment of theelectrophysiology chamber to initiate capture of the helicase-DNAcomplexes in the lysenin nanopore (to give final concentrations ofDNA=0.3 nM, Enzyme=100 nM). Another control was nm at +120 mV for 10mins. Helicase ATPase activity was initiated as required by the additionof NTP (1 mM ATP) to the cis compartment. Experiments were carried outat a constant potential of +120 mV.

Results and Discussion

The addition of Helicase-DNA substrate toLysenin-E92N/E94N/E97N/D121N/D126N (SEQ ID NO: 2 with the mutationsE92N/E94N/E97N/D121N/D126N) as shown in FIG. 1 produces characteristiccurrent blocks as shown in FIG. 5. DNA without helicase bound interactstransiently with the nanopore producing short-lived blocks in current(<<1 second). DNA with helicase bound and active (i.e. moving along theDNA strand under ATPase action) produces long characteristic blockslevels with stepwise changes in current as shown in FIG. 5. DifferentDNA motifs in the nanopore give rise to unique current block levels. Fora given substrate, we observe a characteristic pattern of currenttransitions that reflects the DNA sequence (examples in FIG. 5). Theevent range was observed to be approximately 60 pA.

Example 5

This Example illustrates the use of a Hel308 helicase (Hel308 MBu, SEQID NO: 15) to control the movement of intact DNA strands through amutant lysenin nanopore (Lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A, SEQ IDNO: 2 with mutations E84Q/E85K/E92Q/E97S/D126G/E167A). The generalmethod and substrate employed throughout this Example is shown in FIG. 1and described in the figure caption.

Electrical measurements were acquired as described in Example 2. Afterachieving a single lysenin-E84Q/E85K/E92Q/E97S/D126G/E167A (SEQ ID NO: 2with mutations E84Q/E85K/E92Q/E97S/D126G/E167A) pore in the bilayerunder buffered conditions (625 mM KCl, 100 mM Hepes pH 8.0, 75 mMpotassium ferrocyanide(II), 25 mM potassium ferricyanide(III)), MgCl₂(10 mM) was added to the cis compartment and a control was run at +120mV for 5 mins. Of the 6 mutations, the first 5(E84Q/E85K/E92Q/E97S/D126G) are made within the region of 44 to 126 inaccordance with the invention. The last (E167A) is an additionalmutation outside the region as discussed above. DNA polynucleotide (SEQID NO: 13 and 14) and Hel308 Mbu (SEQ ID NO: 15) were added to 50 μL ofbuffer (625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassiumferrocyanide(II), 25 mM potassium ferricyanide(III), 10 mM MgCl₂) andpre-incubated for 5 mins (DNA=12 nM, Enzyme=2 μM). This pre-incubationmix was added to 950 μL of buffer (625 mM KCl, 100 mM Hepes pH 8.0, 75mM potassium ferrocyanide(II), 25 mM potassium ferricyanide(III), 10 mMMgCl₂) in the cis compartment of the electrophysiology chamber toinitiate capture of the helicase-DNA complexes in the lysenin nanopore(to give final concentrations of DNA=0.6 nM, Enzyme=100 nM). Anothercontrol was run at 120 mV for 10 mins. Helicase ATPase activity wasinitiated as required by the addition of NTP (1 mM ATP) to the ciscompartment. Experiments were carried out at a constant potential ofeither +120 or +180 mV.

Results and Discussion

The addition of Helicase-DNA substrate to lysenin nanoporeLysenin-E84Q/E85K/E92Q/E97S/D126G/E167A (SEQ ID NO: 2 with mutationsE84Q/E85K/E92Q/E97S/D126G/E167A) as shown in FIG. 1 producescharacteristic current blocks as shown in FIG. 6 (at an appliedpotential of +180 mV). DNA without helicase bound interacts transientlywith the nanopore producing short-lived blocks in current (<<1 second).DNA with helicase bound and active moving along the DNA strand underATPase action) produces long characteristic blocks levels with stepwisechanges in current as shown in FIG. 6. Different DNA motifs in thenanopore give rise to unique current block levels.

For a given substrate, we observe a characteristic pattern of currenttransitions that reflects the DNA sequence (examples in FIG. 6). Theevent range was observed to be approximately 30 pA (at an appliedpotential of +180 mV).

Example 6

This Example illustrates the use of a Hel308 helicase (Hel308 MBu, SEQID NO: 15) to control the movement of intact DNA strands through anumber of different mutant lysenin nanopores (see Table 4 for the listof mutant pores tested). The general method and substrate employed inthis Example is shown in FIG. 1 and described in the figure caption.

Electrical measurements were acquired as described in Example 2. Afterachieving a single lysenin mutant pore in the bilayer (see list ofnanopores tested below) under buffered conditions (625 mM KCl, 100 mMHepes pH 8.0, 75 mM potassium ferrocyanide(II), 25 mM potassiumferricyanide(III)), MgCl₂ (10 mM) was added to the cis compartment and acontrol was run at +120 mV for 5 mins. DNA polynucleotide (SEQ ID NO: 13and 14) and Hel308 Mbu (SEQ ID NO: 15) were added to 50 μL of buffer(625 mM KCl, 100 mM Hepes pH 8.0, 75 mM potassium ferrocyanide(II), 25mM potassium ferricyanide(III), pH 8.0, 10 mM MgCl₂) and pre-incubatedfor 5 mins (DNA=12, 6 or 3 nM, Enzyme=2 μM). This pre-incubation mix wasadded to 950 μL of buffer (625 mM KCl, 100 mM Hepes pH 8.0, 75 mMpotassium ferrocyanide(II), 25 mM potassium ferricyanide(III), pH 8.0,10 mM MgCl₂) in the cis compartment of the electrophysiology chamber toinitiate capture of the helicase-DNA complexes in the lysenin nanopore(to give final concentrations of DNA=0.6, 0.3 or 0.15 nM, Enzyme=100nM). Another control was run at +120 mV for 10 mins. Helicase ATPaseactivity was initiated as required by the addition of NTP (1 mM ATP) tothe cis compartment. Experiments were carried out at a constantpotential of either +120 mV or +180 mV.

Results and Discussion

The addition of Helicase-DNA substrate (SEQ ID NO: 13 and 14) to singlelysenin nanopores (see list of pores in Table 4 below) as shown in FIG.1 produces characteristic current blocks as shown in FIGS. 7-12 (at anapplied potential of +120 or +180 mV). DNA without helicase boundinteracts transiently with the nanopore producing short-lived blocks incurrent (<<1 second). DNA with helicase hound and active (i.e. movingalong the DNA strand under ATPase action) produces long characteristicblocks levels with stepwise changes in current as shown in FIG. 7-12 forthe various lysenin mutants tested. Different DNA motifs in the nanoporegive rise to unique current block levels.

TABLE 4 Open pore I (applied Applied potential Helicase- Potential forMutant (SEQ ID NO: 2 in controlled Helicase DNA with the mutations Porebrackets) DNA experiments Conc. Range listed in brackets) insertion (pA)movement (mV) (nM) (pA) Lysenin-(E85K) Yes +277/−186 Not / / / (+/−120mV) investigated Lysenin-(E76S) Yes +283/−188 Not under +120 0.6 /(+/−120 mV) conditions tested Lysenin-(E167A) Yes +305/−198 Not under+120 0.6 / (+/−120 mV) conditions tested Lysenin-(D35Q) Yes +303/−157Not under +120 0.6 / (+/−120 mV) conditions tested Lysenin-(D126Q) Yes+302/−196 Not under +120 0.3 / (+/−120 mV) conditions testedLysenin-(E92N) Yes +260/−130 Yes +120 0.3 / (+/−120 mV) Lysenin-(D121N)Yes +299/−194 Not under +120 0.3 / (+/−120 mV) conditions testedLysenin-(E97N) Yes +308/−199 Not under +120 0.3 / (+/−120 mV) conditionstested Lysenin-(E128N) Yes +295/−188 Not under +120 0.3 / (+/−120 mV)conditions tested Lysenin-(E94N) Yes +151/−275 Not / / / (+/−120 mV)investigated Lysenin- Yes +260/−135 Yes +120 0.6 ~30(E92N/E94N/E97N/D121N/ (+/−120 mV) D126N/E128N) Lysenin- Yes +200/−150Yes (see +180 0.6 ~20 (E76S/E84Q/E85K/E92Q/ (+/−120 mV) FIG. 7)E97S/D126G/E167A) Lysenin- Yes +217/−138 Yes (see +120 0.3 ~20(E84Q/E85K/E92Q/E97S/ (+/−120 mV) FIG. 8) D126G/E167A/E50S) Lysenin- Yes+223/−144 Yes (see +180 0.3 ~25 (E84Q/E85K/E92Q/E97S/ (+/−120 mV) FIG.9) D126G/E167A/E71S) Lysenin- Yes +220/140 Yes +180 0.3 /(E84Q/E85K/E92Q/E97S/ (+/−120 mV) D126G/E167A/E94S) Lysenin- Yes+222/−135 Yes +180 0.3 / (E84Q/E85K/E92Q/E97S/ (+/−120 mV)D126G/E167A/E102S) Lysenin- Yes +226/−139 Yes (see +180 0.6 ~20(E84Q/E85K/E92Q/E97S/ (+/−120 mV) FIG. 10) D126G/E167A/E128S) Lysenin-Yes +249/−153 Yes +120 0.3 ~40 (E84Q/E85K/E92Q/E97S/ (+/−120 mV)D126G/E167A/E135S) Lysenin- Yes +207/−159 Yes (see +120 0.3 ~10(E84Q/E85K/E92Q/E97S/ (+/−120 mV) FIG. 11) D126G/E167A/D68S) Lysenin-Yes +200/−159 Yes (see +120 0.6 ~15 (E84Q/E85K/E92Q/E97S/ (+/−120 mV)FIG. 12) D126G/E167A/D121S) Lysenin-(E97S) Yes +493/−269 Not under +1800.15 / (+/−180 mV) conditions tested Lysenin-(E84Q/E85K) Yes +407/−250Not under +180 0.15 / (+/−180 mV) conditions tested Lysenin- Yes+375/−184 Yes +180 0.15 / (E84Q/E85K/E92Q/D126G/ (+/−180 mV) E167A)Lysenin- Yes +364 Yes +180 0.15 ~12 (E84Q/E85K/E92Q/E97S/ (+180 mV)D126G) Lysenin- Yes +372 Yes +180 0.15 / (E84Q/E85K/E92Q/E97S/ (+180 mV)E167A) Lysenin- Yes +405/−249 Yes +180 0.15 / (E84Q/E85K/E97S/D126G/(+/−180 mV) E167A) Lysenin- Yes +448/−190 Yes +180 0.15 ~15(E85K/E92Q/E97S/D126G/ (+/−180 mV) E167A) Lysenin- Yes +380 Yes +1800.15 ~10 (E84Q/E92Q/E97S/D126G/ (+180 mV) E167A) Lysenin- Yes +222 Yes+120 0.15  ~8 (R52S/E84Q/E85K/E92Q/ (+120 mV) E97S/D126G) Lysenin- Yes+382/−194 Yes +180 0.15 / (N48S/E84Q/E85K/E92Q/ (+180 mV) E97S/D126G)Lysenin- Yes +366 Yes +180 0.15 ~16 (N46S/E84Q/E85K/E92Q/ (+180 mV)E97S/D126G) Lysenin- Yes +369/−184 Not under +180 0.3 /(M44S/E84Q/E85K/E92Q/ (+120 mV) conditions E97S/D126G) tested

Example 7

This example describes a method of synthesising mutant lysenin nanoporesby utilizing E. coli expression.

Materials and Methods

E. coli Rosetta2(DE3)pLysS cells were transformed with a plasmidcontaining the construct Strep-TrxEco-TEV-Lysenin, expression wasinduced with the addition of 0.2 mM IPTG and left overnight at 18° C.The cells were pelleted at 400 rpm for 30 minutes. The cell pellet wasresuspended in 1× Bugbuster in 50 mM Tris 300 mM NaCl 0.1 μl/mlbenzonase 10 μl/ml Calbiochem set V protease inhibitors and left at 4°C. for 4 hours. The lysate was spun at 20000 rpm for 30 minutes andpassed through 0.2 μm filter.

The filtered lysate was loaded onto a StrepTrap column and eluted in 100m114 Tris 300 mM. NaCl 10 mM dethiobiotin pH 8.0. The Strep-TrxEco-TEVtag was removed by incubation with Strep tagged TEV protease (1:20 w/w)at 4° C. overnight. Any uncleaned protein, the cleaved tag and TEVprotease were removed by incubation with strep beads. The beads wereremoved by centrifugation and Sphingomyelin (1 mg/ml) was added to thesupernatant and left overnight at 37° C.

The invention claimed is:
 1. A lysenin pore forming a barrel through amembrane, the lysenin pore comprising at least one lysenin monomer thatcomprises a barrel-forming region comprising the amino acid sequence asset forth in positions 44 to 126 of SEQ ID NO: 2 with one or more aminoacid substitutions, wherein at least one of the amino acid substitutionsintroduces a cysteine, lysine, or non-natural amino acid that is furtherattached to (i) a polyethylene glycol, a nucleic acid, a dye, afluorophore, or a chromophore; (ii) a molecular adaptor; or (iii) apolynucleotide binding protein.
 2. The lysenin pore according to claim1, wherein the one or more amino acid substitutions (a) alter the stericeffect of the monomer, (b) alter the net charge of the monomer, (c)alter the ability of the monomer to hydrogen bond with a polynucleotide,(d) introduce or remove chemical groups that interact throughdelocalized electron pi systems and/or (e) alter the structure of themonomer.
 3. The lysenin pore according to claim 2, wherein (i) the oneor more amino acid substitutions increase or decrease net positivecharge; (ii) net positive charge is increased by introducing one or morepositively charged amino acids and/or neutralising one or more negativecharges; or (iii) one or more negative charges are neutralised bysubstituting one or more negatively charged amino acids with one or moreuncharged amino acids, non-polar amino acids and/or aromatic amino acidsor by introducing one or more positive charged amino acids adjacent toone or more negatively charged amino acids.
 4. The lysenin poreaccording to claim 1, wherein the one or more substitutions comprisesubstitution at one or more of the following positions of SEQ ID NO: 2:M44, N46, N48, E50, R52, H58, D68, F70, E71, S74, E76, S78, Y79, S80,H81, S82, E84, E85, S86, Q87, S89, M90, E92, E94, E97, E102, H103, T104,T106, R115, Q117, N119, D121 and D126.
 5. The lysenin pore according toclaim 4, wherein the amino acid(s) substituted into the one or morepositions are selected from asparagine (N), serine (S), glutamine (Q),arginine (R), glycine (G), tyrosine (Y), aspartic acid (D), leucine (L),lysine (K) or alanine (A).
 6. The lysenin pore mutant lysenin monomeraccording to claim 1, wherein the one or more substitutions comprise oneor more of the following amino acid substitutions: (a) serine (S) atposition 44; (b) serine (S) at position 46; (c) serine (S) at position48; (d) serine (S) at position 52; (e) serine (S) at position 58; (f)serine (S) at position 68; (g) serine (S) at position 70; (h) serine (S)at position 71; (i) serine (S) at position 76; (j) serine (S) atposition 79; (k) serine (S) at position 81; (l) serine (S), asparticacid (D) or glutamine (Q) at position 84; (m) serine (S) or lysine (K)at position 85; (n) serine (S) at position 87; (o) serine (S) atposition 90; (p) asparagine (N) or glutamine (Q) at position 92; (q)serine (S) or asparagine (N) at position 94; (r) serine (S) orasparagine (N) at position 97; (s) serine (S) at position 102; (t)serine (S) at position 103; (u) asparagine (N) or serine (S) at position121; (v) serine (S) at position 50; (w) asparagine (N) or serine (S) atposition 94; (x) asparagine (N) or serine (S) at position 97; (y) serine(S) or asparagine (N) at position 121; (z) asparagine (N) or glutamine(Q) or glycine (G) at position 126; and (aa) serine (S) or asparagine(N) at position
 128. 7. The lysenin pore according to claim 6, whereinthe one or more substitutions comprises amino acid substitutionsselected from the group consisting of: i. one or more of E84D and E85K;ii. one or more of E84Q, E85K, E92Q, E97S, D126G and E167A; iii. one ormore of E92N, E94N, E97N, D121N and D126N; iv. one or more of E92N,E94N, E97N, D121N, D126N and E128N; v. one or more of E76S, E84Q, E85K,E92Q, E97S, D126G and E167A; vi. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E50S; vii. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E71S; viii. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E94S; ix. one or more of E84Q, E85K, E92Q, E97S, D126G,E167A and E102S; x. one or more of E84Q, E85K, E92Q, E97S, D126G, E167Aand E128S; xi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A andE135S; xii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A andD68S; xiii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A andD121S; xiv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167 A andD134S; or xv. one or more of E84D, E85K and E92Q; xvi. one or more ofE84Q, E85K, E92Q, E97S, D126G and E135S; xvii. one or more of E85K,E92Q, E94S, E97S and D126G; xviii. one or more of E76S, E85K, E92Q, E97Sand D126G; xix. one or more of E71S, E85K, E92Q, E97S and D126G; xx. oneor more of D68S, E85K, E92Q, E97S and D126G; xxi. one or more of E85K,E92Q, E97S and D126G; xxii. one or more of E84Q, E85K, E92Q, E97S, H103Sand D126G; xxiii. one or more of E84Q, E85K, M90S, E92Q, E97S and D126G;xxiv. one or more of E84Q, Q87S, E85K, E92Q, E97S and D126G; xxv. one ormore of E84Q, E85S, E92Q, E97S and D126G; xxvi. one or more of E84S,E85K, E92Q, E97S and D126G; xxvii. one or more of H81S, E84Q, E85K,E92Q, E97S and D126G; xxviii. one or more of Y79S, E84Q, E85K, E92Q,E97S and D126G; xxix. one or more of F70S, E84Q, E85K, E92Q, E97S andD126G; xxx. one or more of H58S, E84Q, E85K, E92Q, E97S and D126G; xxxi.one or more of R52S, E84Q, E85K, E92Q, E97S and D126G; xxxii. one ormore of N48S, E84Q, E85K, E92Q, E97S and D126G; xxxiii. one or more ofN46S, E84Q, E85K, E92Q, E97S and D126G; xxxiv. one or more of M44S,E84Q, E85K, E92Q, E97S and D126G; xxxv. one or more of E92Q and E97S;xxxvi. one or more of E84Q, E85K, E92Q and E97S; xxxvii. one or more ofE84Q and E85K; xxxviii. one or more of E84Q, E85K and D126G; xxxix. oneor more of E84Q, E85K, D126G and E167A; xl. one or more of E92Q, E97Sand D126G; xli. one or more of E84Q, E85K, E92Q, E97S and D126G; xlii.one or more of E84Q, E85K, E92Q, E97S and E167A; xliii. one or more ofE84Q, E85K, E92Q, D126G and E167A; xliv. one or more of E84Q, E85K,E97S, D126G and E167A; xlv. one or more of E84Q, E92Q, E97S, D126G andE167A; xlvi. one or more of E85K, E92Q, E97S, D126G and E167A; xlvii.one or more of E84D, E85K and E92Q; xlviii. one or more of E84Q, E85K,E92Q, E97S, D126G, E167A and D121S; xlix. one or more of E84Q, E85K,E92Q, E97S, D126G, E167A and D68S; l. one or more of E84Q, E85K, E92Q,E97S, D126G, E167A and E135S; li. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E128S; lii. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E102S; liii. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E94S; liv. one or more of E84Q, E85K, E92Q, E97S,D126G, E167A and E71S; lv. one or more of E84Q, E85K, E92Q, E97S, D126G,E167A and E50S; lvi. one or more of E76S, E84Q, E85K, E92Q, E97S, D126Gand E167 A; lvii. one or more of E92N, E94N, E97N, D121N, D126N andE128N; lviii. one or more of E92N, E94N, E97N, D121N and D126N; or lix.one or more of E84Q, E85K, E92Q, E97S, D126G and E167A.
 8. The lyseninpore according to claim 7, wherein the one or more substitutionscomprises all of the substitutions in any one of i to lix.
 9. Thelysenin pore according to claim 1, wherein the pore is a homo-oligomericpore.
 10. The lysenin pore according to claim 1, wherein the pore is ahetero-oligomeric pore.
 11. The lysenin pore according to claim 1,wherein the cysteine, lysine, or non-natural amino acid is attached to(i), (ii), or (iii) via a linker.